The document provides instructions for compiling a complete lab report from four biology labs on genomic databases, primer design, PCR, and molecular cloning. It outlines the necessary sections for the report, including an introduction describing the overall question and background, materials and methods, results with data and figures, and a discussion/conclusion section. It also provides additional details on designing a transgene reporter gene based on knowledge gained from the lab exercises, including defining a transgene, necessary gene elements, and ideas for using the transgene.

1. one complete report from all the 4 labs
biology report and need the explanation and answer to help me learn.
Exercises Completed in Module I
Introduction to genomic databases and BLAST: Why do we use BLAST? What does the
output of BLAST searches tell us?
Primer Design and PCR: What is the object of designing good
primers? How will you use the primers?
Molecular Cloning: Why do we clone genes? What do you need to
clone a gene and amplify it?
You have been compiling the “Results” of your report. Let’s put it all
together.
Group Research Report
1) Introduction: Describe the major question, problem, or technical issue addressed. Explain
relevance and background information, including previous work.
2) Materials/Methods: Summarize the materials and methods used to test the hypothesis
for an
assigned gene.
3) Results: Explain the results and how they were obtained. Refer specifically to data in
figures
and/or tables. You will be trained to prepare scientific illustrations for images or
quantitative
data. This section will discuss the methods used (refer to the Methods section of the report).
4) Conclusions and Discussion: Briefly conclude findings and raises future questions.
Summarize
and explain the significance of the work presented in the paper. When appropriate, you are
encouraged to discuss other relevant approaches or articles from the literature.
Discussion
Discussion: Based on the knowledge you have built from the lab
exercises, include a discussion on building a reporter gene or a
transgene: a) define a transgene; b) discuss what gene elements are
necessary for gene expression of artificial genes; c) discuss ideas for
how to use the transgene (see articles posted in Module II in Canvas).
so the report should be a complete report from all 4 labs results. it should have :

2. Introduction ( for all 4 labs results) and ( see the instructions for all 4 labs)
methods (for all 4 labs results) and ( see the instructions for all 4 labs)
Results (Data) (for all 4 labs results) make sure to include table, graph and ( see the
instructions for all 4 labs)
Discussion/Conclusion ( for all 4 labs results) and ( see the instructions for all 4 labs)
Reference(for also 4 labs results)
Grammer
Requirements: depends | .doc file
Biology 366LLab Report Due First Meeting Week 7
Exercises Completed in Module I•Introduction to genomic databases and BLAST: Why do
we use BLAST? What does the output of BLAST searches tell us?•Primer Design and PCR:
What is the object of designing good primers? How will you use the
primers?•MolecularCloning:Why do we clone genes? What
doyouneedtocloneageneandamplifyit?•You have been compiling the “Results” of your
report. Let’s put it all together.
Group Research Report1)Introduction: Describe the major question, problem, or technical
issue addressed. Explain relevance and background information, including previous
work.2)Materials/Methods: Summarize the materials and methods used to test the
hypothesis for an assigned gene.3)Results: Explain the results and how they were obtained.
Refer specifically to data in figures and/or tables. You will be trained to prepare scientific
illustrations for images or quantitative data. This section will discuss the methods used
(refer to the Methods section of the report).4)Conclusions and Discussion: Briefly conclude
findings and raises future questions. Summarize and explain the significance of the work
presented in the paper. When appropriate, you are encouraged to discuss other relevant
approaches or articles from the literature.
Discussion•Discussion: Based on the knowledge you have built from the lab exercises,
include a discussion on building a reporter gene or a transgene: a) define a transgene; b)
discuss what gene elements are necessary for gene expression of artificial genes; c) discuss
ideas for how to use the transgene (see articles posted in Module II in Canvas).
Gene expression in living cells is often difficult to detect because of limited access of
substrates to marker enzymes. Here gene expression in specific neurons of the nematode
Caenorhabditis elegans is monitored by the bright green fluorescence of the green
fluorescent protein (GFP) from the jellyfish Aequorea victoria. The GFP fills entire neurons,
including in one neuron an extended, fanned growth cone visible in the tail end (upper
portion) of the nematode. See page 802. [Photo: Martin
Chalfie]https://www.nobelprize.org/prizes/chemistry/2008/summary/
Group No.:Biology 366L: Spring 2023Grader:Laboratory Report Rubric Total Points
100ExcellentGoodFairNeeds ImprovementIntroduction (26 pts)1. Clearly communicates
the question that is trying to be answered. 2. States a clear a goal or
hypothesis that is predictable and testable. 3.

3. Provides evidence to support hypothesis from background/research. 4.
Incorporated edits to improve introduction from previous report.Some of the "excellent"
items are missing.Methods (8 pts)A description or step-by-step list of how the experiment
was performed. Description, unclear could not be repeated.Results (Data) (26 pts)1. Results
are clearly recorded and organized. 2. Figures are of publish
quality and clearly labeled and easy to follow with written results.
3. Easy for the reader to see trends in the results. 4. Clearly
transitioned and incorporated new data into results.One of the "excellent" items is
missing.Two of the "excellent" items are missing.Three of the "excellent" items are
missing.Discussion/Conclusion (28 pts)1. Summarizes essential data used to draw
conclusions. 2. Discusses application and
implications of results. ("real world connections"). 3. Puts findings
into context of previously published results. 4. Describes future
direction of project. 5. Concludes with an overall statement (e.g., In
summary, this work...). One of the "excellent" items is missing.Two of the "excellent"
items are missing.Three of the "excellent" items are missing.References (6 pts)Clearly
organized.Missing appropriate citations.Format and Grammar (6 pts) Neat organized with
clear headings, few grammatical and spelling errors.Somewhat lacking in organization,
multiple spelling and grammar errors, not neat.Comments:
Transgenes –definition, design and testing
A Typical Eukaryotic GeneThe typical eukaryotic gene is composed of a cis-regulatory
domain as well as the sequences required to encode the protein Start of
transcriptionExons/intronsCis-regulatory region
The cis-regulatory domain contains binding sites for transcription factors –the binding of
these proteins regulates gene expression
In multicellular organisms, genes can be expressed in different cell types (like the
Cionaembryos to the left –green color), at different times and at different levelsThe cis-
regulatory sequences of the gene regulate temporal, spatial and quantitative expression
A transgene combines these cis-regulatory sequences with a reporter gene (like GFP). When
expressed in the cell/tissue/organism it should reflect the expression of the endogenous
gene.To know if your transgene works correctly, you need to have characterize the
expression of the endogenous gene you are interested in studying: i.e. its spatial, temporal
and quantitative expression. This can be analyzed using in situ hybridization (see next
slide).You then generate a transgenic embryo and analyze the expression of the transgene.
If it matches the expression pattern of the endogenous gene, then you have likely captured
the relevant regulatory information on the piece of cis-regulatory sequence you used in
your transgene.
In situ hybridization can be used to localize gene expression in cells, tissues and
organismsThe purple stain shows you where the gene is expressed
Steps to design/test a transgene:•Start with genome sequence 5’ to the start of the gene of
interest –why? This is commonly where most regulatory sequence information
exists•Identify appropriate restriction enzymes to use to clone the DNA fragment•Create a
translation fusion between exon of the gene and the reporter protein –why? Because

4. studies have shown that including an intron in your transgene can enhance its expression.
There can also be regulatory elements located in introns•Express your transgene in your
organism. If it is expressed the same as the exogenous gene, you most likely have all of the
required regulatory elements contained within your fragment. If not, you are missing
important regulatory regions and must locate those. They could be in other introns,
downstream of the gene of further upstream. You would need to design and test additional
transgene reporters.Your goal for this exercise is to design a transgene reporter for the gene
your group has been working on.
Go to ensemble.org and find the Brachyury gene as you have done beforeHere we want to
grad sequence upstream (i.e. 5’) of the gene. The arrow in the name is pointing to the right
so we are in the correct orientation. Click and drag to zoom in a bit more.
Brachyury geneUpstream geneWe want sequence from this region, type the values above to
zoom inType in range 3900-6600
Click on export data to get a fasta-formatted sequence of this region.Note, if your gene is
oriented in the opposite direction, you can still follow these instructions to get your
sequence. In Snapgeneyou can easily reverse your sequence to work with it.
Hit next, then select “text” that will give you the sequence you see to the right. Grab that
sequence…
Using Snapgene, add this sequence as a new sequencePut the sequence in “sequence view”,
we are going to annotate the first couple of coding exons to help us design our transgene.
In ensembl, pull up the Brachyury exon sequencesWe just need to first two exons, copy the
first exon sequence (just the blue sequence is ok)
In Snapgene, navigate to align 2 sequences
The Brachyury sequence you grabbedThe first exon sequence you just copiedAlign them!
Note the endpoints, then let’s annotate the sequence
Select the same region of sequence (2394-2462) then click on add features-> add feature
Name itExon or cds, once you hit translate it will force it to be cdsClick on feature
translation options to select the proper reading frame. This is correct –hint you get compare
to the Brachyury protein sequence to confirm.
Now do the same steps from exon 2Now both exons are annotated and you have the correct
reading frames aligned with you sequence. This will help you with getting the restriction
enzyme site in the proper reading frame.
This is the transgene cloning vectorIt has the following parts: AmpR(selectable marker)Ori
–plasmid origin of replicationMCS –multiple cloning siteCrHistone–histone coding
regionYFP –YFP coding regionCrBetaActin3’ UTR –3’UTRYou will be cloning your genome
fragment into the MCS and cloning it in-frame with the histone coding region. This will
produce a reporter transgene that will express YFP in the nuclei of cells (because YFP is
fused to a histone)
Go to sequence viewNow you can see the enzymes in the polylinkerand the reading frame
relative to the histone sequence (blue box).Now you want to go the Brachyury sequence in
Snapgeneand see if it has any of the polylinkersequences. If it does, skip those and make
note of which polylinkerenzymes you could use to clone your transgene. Note that both

5. PvuIIand BamHIare not in bold, indicating these are not unique sites –don’t use them!To
save you some time, you can go to enzymes-> noncuttersand see a list of enzymes that don’t
cut your sequence (you can export this information to make it easier to search)
These PL enzymes do not cut and are OK to
use:AatIINotIXhoIHindIIISphIPstISalISmaIAcc65iThese enzymes that you can use are listed
from the 5’ to the 3’ of the MCSIf you look at the MCS, you’ll see a stop codon present 5’ of
the HindIIIsite. Therefore, make sure your 3’ most enzyme is between HindIIIand Acc65i.
Let’s use XhoIfor the 5’ end of DNA fragment and Acc65i for the 3’ end of the fragment
Start with the 5’ (easy) endGrab the first 25-30 nucleotides and check against the NEB Q5
DNA polymerase calculator from last labhttps://tmcalculator.neb.com/#!/mainNow add a
few ntplus your RE site for XhoI:5’ (forward) primer:
gcgctcgagCTTCTCATAGAAATACAATACAAGTTACG
The 3’ primer is a bit more complex….You can hover the mouse over the RE site to
determine the sequence and see how it lines up with the coding region.For Acc65i, the site
is GGATCC and this is the reading frame that we to maintain:cgGGTA CcaR V PWhat we
want to do in this case is to find a region in exon 2, find a codon with “G” in position 3 and
then add GTACCgcgto add our RE
Here we use this methionineGATCCAACTGCGATGTATTCCGTCATG gtaccgcg
Add your primers to the Brachyury sequence
Add your primers to the Brachyury sequenceDon’t forget to reverse compliment the reverse
primer!
Now let’s build the reporterUse your primers to generate a PCR fragment of the genomic
region you want to cloneUsing this fragment, subcloneit into the transgene vector with
XhoIand Acc65iUse the same steps you did previously
The resulting clone, check the sequence view.
Here is where your 3’ end of your clone went into the vectorNotice that the reading runs
from exon 2 into the histone sequence without frameshifts
The sequence you just cloned corresponds the region known to be an active regulatory
domain for the CionaBrachyury gene.If you put this transgene into the ascidian Ciona, the
cells where Brachyury is expressed (the notochord cells) would be expressing the transgene
as shown in an image of living embryos above.
AssignmentUsing the Cionagene assigned to your group from last week, build a transgene
following the example in these slidesDocument your steps, screen grabs can be used if
needed:1)What region did you select (like slide #9).2)Show your sequence annotation (like
slide #20). If your gene only has one exon, just use that single exon3)Indicate which
enzymes you can use (like slide 23). If you can’t identify a pair of enzymes, what could you
do?4)Show your forward and reverse primers (like slides 24 and 26)5)Show your
completed transgene (slide 30)6)Show a closeupof the 3’ cloning region indicating that you
cloned your sequence in-frame with the reporter construct (like slide 31).
The Polymerase Chain Reaction (PCR) and PCR-based cloning•PCR has revolutionized the
way in which nucleic acids can be identified, amplified and used in molecular biology
Key milestones in the development of PCRhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcrWatson and CrickNobel Prize

6. for DNA structureArthur and SylvyKornbergNobel Prize for DNA replicationHar
KhoranaNobel Prize for genetic codeKleppe, a postdoc of Khorana’s proposed 2 primer
system but never did the experiment!Kary MullisBrock -1969 isolated a thermophile from
Yellowstone NP -Thermus aquaticus. 1976 -Chien and Trelapurified DNA polymerase
showing it was active at 80C, clone reported in 1988Needed to add enzymes each cycle,
needed to move tube by hand to different water baths
•This video provides a brief review of the PCR process:
https://www.youtube.com/watch?v=c07_5BfIDTw
The implementation of PCR required several key developments•Discovery and
characteristic of DNA polymerase enzymes•Generation of oligonucleotides which could be
used as “primers”•Realization that orienting primers opposed to one another could cause
exponential amplification•Isolation and cloning of thermostable DNA polymerase (Taq
polymerase)•Engineering of thermocycler machines that could automatically change
sample temperatures during the process.
Key steps in the PCR cycle: Denaturation, Annealing, Extensionhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcr
Primer design•Although designing primers seems straightforward, there are a variety of
issues that one must think about when planning for an experiment. The following video
outlines some of these issues which we will
discuss.•https://www.youtube.com/watch?v=mcOwlFVEino
Primer design considerations –length, GC content and TmGC content, length and melting
temperature. These are related as follows: longer primers and/or those with higher GC
content have higher melting points. Generally, you want to end the 3’ primer sequence with
a “C” or “G” (what is known as a “GC” clamp). This may require you to move the location of
the primer along your sequence to be amplified. Primers should have a Tm within about 5C
of each other and within the range of 65-75C, this will help to avoid mis-
primingOligonucleotides are synthesized 3’ to 5’ and the longer the primer, the lower the
percentage of full-length product in the synthesis reaction% Full length product =
(efficiency)(n-1) where efficiency is the synthesis coupling efficiency (typically about 99%)
and n is the number of cycles. For a 50 ntlong primer:% full product = (0.99)49= 61%. 49
cycles are needed to make a 50-mer
Primer design considerations –sequence composition•Avoid runs of 4 or more bases
(“AAAA” or “CCCC”)•Avoid dinucleotide repeats (i.e. “ATATATAT”)•Avoid intra-or inter-
primer homology as this will cause the primers to bind internally or with each other
interfering with the amplification of your product.Hairpin from internal bindingInter-
primer homologyLastly, if you plan to incorporate restriction enzymes sites for cloning, add
those with 3-5 bp additional sequence to allow efficient enzyme activity
Given the previous constraints, software packages have been developed to assist with
primer designIn this exercise we will be amplifying specific regions of genes and this
typically requires one to hand-design primers sequences. We will use a tool called
PrimerBlastto help us determine what the best design would be.First we will design primer
sets to specifically amplify the coding region of a gene so that it can be cloned into an
expression vector with a specific reading frame.Second, we will work to design primers to

7. make a transgene reporter.The following slides will walk you through the process.
Sequences to useFor simplicity, you will be working with several different sequences from
the ascidian Cionaintestinalis. Recall that you already have worked with Cionasequences at
ensemble, here you will be provided with community-annotated transcripts that include 5’
and 3’ UTRs.The cloning vectors will also be explained and annotatedFirst, let’s translate the
BrachyurycDNA sequence into the correct reading frame.
Use https://web.expasy.org/translate/to translate Make sure you select:the bottom radio
buttonForward strandThen hit translate
Scrolling down shows frame 2 as the correct reading
framecacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgt
catcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTP
PSLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonHINT –put
your sequences in font Courier New so everything aligns
We want to design PCR primers that will amplify the entire open reading frame from the
start to the stop
codoncacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacg
tcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonLet’s make a first pass at making a primer. Grab about 7-8 codons of sequence,
stopping at position 2 of the last codon. Why should you stop there?
Check your primer sequence for use with Q5 DNA polymerase (a common PCR enzyme)
https://tmcalculator.neb.com/#!/mainLooks good, now do the second primer
atgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSLtga
cgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonThese look good, let’s
check them for other characteristics
Go to Primer Blast: https://www.ncbi.nlm.nih.gov/tools/primer-blast/BrachyurycDNA
sequenceForward primerReverse primerFor your reverse primer use this reverse-
compliment tool: https://www.bioinformatics.org/sms2/rev_comp.htmlUse these values
Scroll down, remove humans and add CionaintestinalisThis will allow the software to check
your primer design against the genome to help identify off-target hitsHit “get primers” and
wait for search to complete.
Output –not bad, but some 3’ and self complimentarityForward: atgttttcgaagctcagtgcgag3’
self comp, can form hairpin in primerPoly T run, sometimes you can’t avoidClick here for
detailed results
Tweak the forward
primercacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgac
gtcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonNew forward: atgttttcgaagctcagtgc. This is shorter so the Tm decreases, make the
reverse primer shorter
tooatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSL
tgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtga
Primers look good, let’s do a virtual PCR

8. PCR test:
https://www.bioinformatics.org/sms2/pcr_products.htmlBrachyurysequenceForward and
reverse primers (make sure to reverse compliment the reverse primer)
PCR Products results –looks like the correctregion>1359 bpproduct from linear template
KH.S1404.1.v1.A.nonSL1-1 Brachyury, base 26 to base 1384 (Forward -Reverse).
ATGTTTTCGAAGCTCAGTGCGAGTTACAAACCTATAATGACGTCATCAGATAGTAAGTTA
GCAGGTATGACGTCATCAGAATCAATTGAAACATGTGCAGTAAAGATGGCGCTAATAGAG
CATGGTTTATGGTCGAGGTTTCACGCGTTTGTCAACGAGATGATTGTGACAAAAAATGGA
CGACGAATGTTTCCGGTTCTTAAAACATCGATTACGGGACTTGATCCAACTGCGATGTAT
TCCGTCATGCTTGACTTTGTACCCGTGGATAACAATAGATGGAAATATGTGAACGGTGAG
TGGATCCCCGGGGGAAAACCCGAACCCCATGTTTCGTCATGTGCTTATATTCACCCCGAT
TCACCCAACTTTGGTTCGCACTGGATGAAACAACCCGTTGGTTTCAGTCGCGTCAAACTT
ACTAATAAAGCTACAGGGAACCCCCAGCAAATAATGTTGAACTCTCTGCATAAATACGAA
CCCAGGATCCACATCATGCGCGTTGGGGGCGCTGAATCGCAACAAGTCGTTGCCTCGCAT
TCATTTCAGGAGACAAGGTTCATTGCTGTTACTGCTTACCAAAACGAAGATGTTACTTCG
CTCAAGATAAAATATAATCCTTTCGCCAAAGCTTTCCTCGACGCAAAGGAGAGCCGCTCA
GGGAGTGAAAATTATTTTAAAGATTCAACGAAAGCGGGTTCGTCGCAAAATTATTCGAGA
GCGAACACATGGACGGCAAATCAAAGCAACCCAACATTCAATCAATGTCAGTATGAATCA
GGGATTCCCCCATTCCATTACCCCATCCCAAACCAACCGAAAACAACAAGAGAGCGGCGC
ATGAGCCGCACACAAAGAAGCCATCCCTACAAACCATCCACAACGCAAATTTATCAAGAT
TTCCAACCAACCAATTACCCCGCCCTACCAAACGACCAATGGCAATCAAGTATCGAAGGT
CATGAACTCGATGAGGGACACTTTAGTCTCGAACCAGTCTCTGTAGATGACGTCACCGCC
TTAGGATTCGACACCCCGCATCAGGGTTTTGCAACAAACGATCTTCTTTCAATAGAGCCT
TCCTATTCGTTAGATTATCCTCAATTCACGACCACGTGGTCCTACCCGACCAATCACATG
CCAGGATATGTTTCAAACAGTCCAATCAGAAGCCTGGAACAACCGGAGTACTTTTATAGA
GGCTATGACGTATCACAGCAAAATTACGTCACGATGACGTCAGCAGATGTTAGTAATGTG
ACGTCATCATTATACGAAACTCCGTCACCGGGGGTCATGCAAAGGCCACAAGAAGATTTT
TCAATCGCGTATACGCCGCTTACGCCACCTTCTTTGTGACheck the ORF at
https://web.expasy.org/translate/
Looks good!
Now let’s clone this sequence into a bacterial expression vector (plasmid) so we can make
BrachyuryproteinReview that plasmids are circular DNA pieces present in bacteria.
Researchers have adapted these molecules for use in molecular BiologyIf you’d like a
reminder on basic cloning approaches, please review these videos:Restriction enzyme
cloning: https://www.neb.com/tools-and-resources/video-library/cloning-with-
restriction-enzymesOur approach: https://www.neb.com/tools-and-resources/video-
library/overview-of-traditional-cloning
Bacterial vector for making proteinsFree viewer at snapgene.com. This will let you do basic
analysis like see where restriction enzymes cutVector = pTrcHisA, B or C
The polylinker(cloning site) has recognition sites for different restriction enzymesWe need
to identify the correct enzymes to use and add those to our PCR primers.
This vector has three versions which changes the reading frame around the restriction
enzyme sitesABCSince we can use PCR to set the reading frame, we can just focus on one

9. vector, so we will use pTrcHisC
Here reading frame is importantBamHITGGATCCGACCTAGGCW
IBglIIGAGATCTGCTCTATGACD LAcc65i/KpnIGGTACCTAACCCTGGTV PRE sites are in
PURPLECodon is in REDTo express our protein, we need to add the correct RE sites to our
primers with the correct reading frameThe enzymes in our polylinkerare: BamHI, BglII, PstI,
Acc65i, EcoRIand HindIIIOur DNA insert must be oriented ATG -> stop in this direction
Let’s see what enzymes we can use: https://nc3.neb.com/NEBcutter/Paste your FASTA
sequence in, preferably from your PCR productOverviewExamine enzyme listLook at zero
cutters –these are enzymes that don’t cut our PCR product
BamHIcuts our DNABglIIdoes not cutPstIdoes not cutAcc65i does not cutEcoRIdoes not
cutHindIIIcuts our DNALet’s use Acc65i and EcoRIThese let us insert our DNA in the proper
orientationThe enzymes in our polylinkerare: BamHI, BglII, PstI, Acc65i, EcoRIand
HindIIIWhat happens if we can’t usable RE sites??
We need to worry about the reading frame for Acc65i on our clone. Because EcoRIis after
the stop codon, reading frame doesn’t matterAcc65i/KpnIGGTACCTAACCCTGGTV
PcacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgtcatc
agatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonOur primer needs to preserve this reading frame: G GTA CCT AOur primer sequence
starts with the start codon ATGIf we keep the prolinecodon from the RE and simply add our
primer sequence we are good to go:G GTA CCT atgttttcgaagctcagtgcgV P M F S K L S
ALastly, add 3 nucleotides to the 5Õ end of the primer so the enzyme can cut: CgcG GTA CCT
atgttttcgaagctcagtgcg
The reverse primer is
easieratgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPP
SLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtgaCttacgccaccttctttgtgaGAATTCgcgEcoRIRemember to reverse
compliment:cgcGAATTCtcacaaagaaggtggcgtaaG
Group assignmentEach group will be given a gene to work with, these are provided
separately. I’ve also provided the Brachyurysequence used in the powerpointwalk-through.
1.For your gene, first identify the ORF as on slide 12. Show the correct start and stop
codons2.Design your first round of primers like slides 13-153.Check your primers in Primer
Blast and report the output (slide 18). If you find there are additional matches, you can
always blast your ntsequence at ncbito identify if you are hitting alternative splice forms of
your gene. If you do, will this interfere with your PCR amplification? Why or why not?
4.Tweak primers if needed and report like slides 19-205.Check your PCR product like on
slides 22 and 236.Check your sequence (the PCR product) on NEBCutterand report the
results (i.e. slides 29/30). Which enzymes can you use for your primers. If you can’t use any
enzymes, what could you do? Hint: sequence mutation would be needed in your PCR
product –what would you need to do?7.Report final forward and reverse primer design
(like slides 31-32)
Website referencesORF finder: https://web.expasy.org/translate/Primer Blast:
https://www.ncbi.nlm.nih.gov/tools/primer-blast/Reverse compliment:
https://www.bioinformatics.org/sms2/rev_comp.htmlNEB Q5 calculator:

10. https://tmcalculator.neb.com/#!/mainNEB Cutter: https://nc3.neb.com/NEBcutterPCR
simulation: https://www.bioinformatics.org/sms2/pcr_products.html
Gene orthologues, genome browsers and gene landmarksIn this week’s lab we will build
upon the exercises from last week (extracting sequences from DBs, BLAST searching,
translating)Here we use a test gene called Brachyury to explore genome browsers and gene
structure These exercises will prepare you for the next sequence exercises –designing
primers to clone and mutate genes and build transgene reporters.
Important Links•Ensembl genome website:
https://uswest.ensembl.org/index.html•Ensembl metazoa:
https://metazoa.ensembl.org/index.html•https://www.youtube.com/watch?v=C2g37X_uM
ok•The above video link provides a walk through on how to browse genomes and genes at
ensembl
Brachyury is a transcription factor important for early development –it will be our example
proteinhttps://en.wikipedia.org/wiki/T-box_transcription_factor_TThis is a model of the
Brachyury protein binding to DNA (purple molecule)
Brachyury belongs to a family of genes called Tbox genes.
https://www.pnas.org/doi/10.1073/pnas.1309748110Brachyury itself is widely
distributed among metazoans and even fungiWe will use this characteristic to learn how to
explore orthologues of Brachyury in different animal genomes.
Select animal genomes –based on a sample of Biology faculty model
organismsDrosophila(fruit fly): Bernstein, Cripps, Capelson
https://uswest.ensembl.org/Drosophila_melanogaster/Info/IndexSea Urchin:
SchrankelPlanarian: ZayasAscidian:
Zellerhttps://metazoa.ensembl.org/Lytechinus_variegatus_gca018143015v1/Info/Indexhtt
ps://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Indexhttps://usw
est.ensembl.org/Ciona_intestinalis/Info/IndexAll of these animals have much smaller
genomes than vertebrates (mice, humans, fish) making some of our exercises easier to do. C.
elegans: Luallenhttps://uswest.ensembl.org/Caenorhabditis_elegans/Info/Index
Let’s get started! Go to ensemble.orgEnsembl maintains genome assemblies for dozens of
organisms and is a one-stop place for doing comparative genome browsing. Your TAs may
use other genome sites for their own research –ask them about it!
Retrieve the brachyury protein from the mouse genome:Select the mouse genomeType
“brachyury” in the box and hit go
Find the circled link and click it
General information, note the accession number (ENSMU…)Position in genome,
chromosome 17,Nucleotides 8653255 to 8661328Forward strandLinks to similar proteins
Mouse Brachyury on the genome browserA genome browser is typically web-based and
shows a variety of information that you can explore to learn about genes and
genomesBefore moving on, let’s explore the browser interface and the information it
displays.Click here!
Mouse Brachyury on the genome browserClick here!
Detailed viewDirection of transcription ->8 exons, 7 introns1234 56785’UTR3’UTRThe rest
of the exons have coding sequenceClick on cDNA after viewing slide

11. cDNA sequenceNote the sequence is colored coded and the translation is also shown
Exon view. Note the exon sequence is shown separately from the intron sequences. The UTR
sequences are also color coded
Protein view.You can download the Fasta formatted protein sequence
Mouse Brachyury on the genome browserGo back to this viewClick on “region in detail”
Chromosome and positionChromosomal region surrounding the Brachyury gene, ~500
MBZoom controls for belowBrachyury gene
Arrows shift view upstream or downstreamThese are called “tracks” and describe various
kinds of data to display such as predicted/known genes, SNPs, regulatory gene information,
etcT-201 is BrachyurypromoterTry zooming outNote that there are several alternative
splice forms of Brachyury
BrachyurypromoterenhancerIf you wanted to build a transgene, you would need to include
promoter/enhancer information
Go back to this page, click on tab labeled Gene: TIf you click on orthologues, you can get
other proteins that match, but we’ll do a Blast instead
Paste in your mouse Fasta file for Brachyury, select Drosophila, C. elegansand Ciona
intestinalis for other species and then run Blast
Paste in your mouse Fasta file for Brachyury, select Drosophilaand Ciona interstinalis for
other species and then run BlastSearch the protein DBs for your species –why? This will
allow you to BLAST against a protein database that will then allow you to find the
corresponding genomic region. BLASTing against the genome will split your hits up among
the various exons of the gene.
Check your results!
For Ciona, the top hit is the CionaBrachyury geneClick on genomic location
Brachyury!You may need to adjust the view to see this image –zoom out move the window
An easy way to get the view you want is to zoom out, then drag a box about the region you
want to focus on. Select jump to region.
Now you have a centered view of the CionaBrachyury gene. Instead of zooming out and
selecting the region, you can just type in the coordinatesThis small > symbol tells you the
direction of transcription
Now look at the Drosophilahit repeating the steps needed to view the gene plotted on the
genome.This small > symbol tells you the direction of transcriptionNote: although the
Brachyury gene is found in a wide group of organisms, it has been lost in C. elegans and in
the Planarian Schmidtea mediterranea.
Compare the three Brachyury gene structuresmouseDrosophilaCionaHow many exons?
How many BP in each genome? Characterize the UTRs/coding regionsAlternative
transcripts
exonexonexonexonexonexonexonExon-intronboundaryHow to identify intron-exon
boundaries. Each line of nucleotides is the same length, so you can easily line them upThis is
the mouse Brachyury gene
Helpful hints:1)Paste your cDNA sequence into a word or google doc. Take a few minutes to
clean up the view so it is easy to read.2)Make sure you put the text into “courier new” font
and adjust the font size until it looks nice3)Now just search for the sequence from the exon

12. view in your document. The first exon boundary ends at “GAACGGCAG”4)You can use the
amino coordinates to help you find the junctions in your clustalw alignment (next slide)
T-201
MSSPGTESAGKSLQYRVDHLLSAVESELQAGSEKGDPTERELRVGLEESELWLRFKELTN60brach
yury-201 --------------------MTSSDSKLAGMTSSESIETCAVKMALIEHGLWSRFHAFVN40::: :*:*
. :.. . :::.* * ** **: :.*T-201
EMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVTADNHRWKYVNGEWVPGGKPEPQAP120
brachyury-201
EMIVTKNGRRMFPVLKTSITGLDPTAMYSVMLDFVPVDNNRWKYVNGEWIPGGKPEPHVS100**
**************..::****.****.:**** .**:*********:*******:. T-201
SCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGG-GQIMLNSLHKYEPRIHIVRVGGP-
178brachyury-201
SCAYIHPDSPNFGSHWMKQPVGFSRVKLTNKATGNPQQIMLNSLHKYEPRIHIMRVGGAE160**.*
*********:**** **.**:****** .*. ****************:**** T-201 -
QRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERNDHKDVMEEPGDCQ237brach
yury-201
SQQVVASHSFQETRFIAVTAYQNEDVTSLKIKYNPFAKAFLDAKERSGSENYFKDSTKAG220*::::**
.* **:**********::*:******************.. :: ::: .. T-201 Q-PGYSQ-WGWLVPGAG---
TLCPPASSHPQFGGSLSLPSTHGCER-YPALRNHRSSPYP291brachyury-201
SSQNYSRANTWTANQSNPTFNQCQYESGIPPFHYPI--PNQPKTTRERRMSRTQRSHPYK278. .**:
* . :. . * *. * * : *. * *.:** ** T-201 SPYAHRNSSPTYADNSSACLSMLQSHDNWSSLG-
----VPGHTSMLPVSHNASP------340brachyury-201 PSTT-----QIYQDFQPTNYP-
ALPNDQWQSSIEGHELDEGHFSLEPVSVDDVTALGFDT332: * * . : :*:*.* ** *: *** :
T-201 -----------------PTGSSQYPSLWSVSNGTITPGSQTAGVS--
NGLGAQFFRGSPA381brachyury-201 PHQGFATNDLLSIEPSYSLDYPQFTTTWSYPTNHMP-
GYVSNSPIRSLEQPEYFYRGYDV391. *: : ** .. : * : . *:** .T-201
HYTPLTHTVSAATSSSSGSPMYEGAATVTDISDSQYDTAQSLLIASWTPVSPPSM436brachyury-
201 SQQNYVTMT-SADVSNVTSSLYETPS------PGVMQRPQEDFSIAYTPLTPPSL439. . :* *. *
:** : . : *. : ::**::***:Just the mouse (top) and Ciona (bottom) sequences are shown.Use
font “courier new” to get your alignment to look nice.In this example, the amino acids
located at the exon-intron borders have been highlightedNote that some boundaries are
conserved(i.e. same relative positions in proteins, red circles)While others are not (blue
circles)
Analysis and questions for the exampleCompare and contrast your three brachyury genes
(mouse, Ciona, Drosophila):Do the genes have the same numbers of exons in each
species?Do the genes span the same amount of space (length) in the genome?Are the 5’ and
3’ UTR regions the same length? Are the UTRs located on their own exon or on an exon
which contains coding region?Are the exons located at the same positions within each gene?
You will need to extract the exon sequence for each gene for this and compare to the cDNA
sequence for eachPerform a clustalw alignment of your 5 proteins and annotate the
positions of the intron/exon boundaries on the alignment (this is on slide 32 for you)
Data needed for assignments•Each group has a gene to analyze in 5 genomes (Drosophila,
Ciona, C. elegans, Sea Urchin and Planarian. These are listed below.•Use the accession

13. number to find the corresponding mouse protein sequence, use that in a blast search•For
Drosophila, C.elegans and Ciona intestinalis: https://uswest.ensembl.org/index.htmlFrom
that site, select blast and input your protein sequence. Be sure to select all three species so
that you only need to do one blast run for these 3 species•For the sea urchin, use this link:
https://metazoa.ensembl.org/Strongylocentrotus_purpuratus/Info/Indexand select blast
from the top menu•For planarian, use this link:
https://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Index/and
select blast from the top menu•Retrieve the protein sequence from each genome and do a
clustalw multiple alignment (we did that last week). This information will be used later as in
the example.•Put screen grabs of the genes for each species in your document (i.e. like on
slide 29)•Retrieve the exon and cDNA sequences for each gene, you will need that to answer
some questions.
If your gene has alternative splice forms, use the longest one with the most exons
Mouse gene assignments by
group1ENSMUSP000000253742ENSMUSP000000411183MGP_WSBEiJ_P00627004ENSMU
SP000000692775ENSMUSP000000060716ENSMUSP000001273967ENSMUSP000000580
208ENSMUSP00000044879
What to turn in:•Compare and contrast your five genes (C. elegans, sea urchin, planarian,
Ciona, Drosophila). Make a figure like on slide 29 and answer the next five questions•Do the
genes have the same numbers of exons in each species?•Do the genes span the same
amount of space (length) in the genome?•Are the 5’ and 3’ UTR regions the same length?
•Are the UTRs located on their own exon or on an exon which contains coding region?•Do
all of the genes have annotated 5’ and 3’ UTRs? If not, why do you think that is?•Perform a
clustalw alignment of your 5 proteins and annotate the positions of the intron/exon
boundaries on the alignment like on slide 32.•Are the exons located at the same positions
within each gene? Why do you think some boundaries are preserved while others are
unique?
Biology 366LLab Report Due First Meeting Week 7
Exercises Completed in Module I•Introduction to genomic databases and BLAST: Why do
we use BLAST? What does the output of BLAST searches tell us?•Primer Design and PCR:
What is the object of designing good primers? How will you use the
primers?•MolecularCloning:Why do we clone genes? What
doyouneedtocloneageneandamplifyit?•You have been compiling the “Results” of your
report. Let’s put it all together.
Group Research Report1)Introduction: Describe the major question, problem, or technical
issue addressed. Explain relevance and background information, including previous
work.2)Materials/Methods: Summarize the materials and methods used to test the
hypothesis for an assigned gene.3)Results: Explain the results and how they were obtained.
Refer specifically to data in figures and/or tables. You will be trained to prepare scientific
illustrations for images or quantitative data. This section will discuss the methods used
(refer to the Methods section of the report).4)Conclusions and Discussion: Briefly conclude
findings and raises future questions. Summarize and explain the significance of the work
presented in the paper. When appropriate, you are encouraged to discuss other relevant

14. approaches or articles from the literature.
Discussion•Discussion: Based on the knowledge you have built from the lab exercises,
include a discussion on building a reporter gene or a transgene: a) define a transgene; b)
discuss what gene elements are necessary for gene expression of artificial genes; c) discuss
ideas for how to use the transgene (see articles posted in Module II in Canvas).
Gene expression in living cells is often difficult to detect because of limited access of
substrates to marker enzymes. Here gene expression in specific neurons of the nematode
Caenorhabditis elegans is monitored by the bright green fluorescence of the green
fluorescent protein (GFP) from the jellyfish Aequorea victoria. The GFP fills entire neurons,
including in one neuron an extended, fanned growth cone visible in the tail end (upper
portion) of the nematode. See page 802. [Photo: Martin
Chalfie]https://www.nobelprize.org/prizes/chemistry/2008/summary/
Group No.:Biology 366L: Spring 2023Grader:Laboratory Report Rubric Total Points
100ExcellentGoodFairNeeds ImprovementIntroduction (26 pts)1. Clearly communicates
the question that is trying to be answered. 2. States a clear a goal or
hypothesis that is predictable and testable. 3.
Provides evidence to support hypothesis from background/research. 4.
Incorporated edits to improve introduction from previous report.Some of the "excellent"
items are missing.Methods (8 pts)A description or step-by-step list of how the experiment
was performed. Description, unclear could not be repeated.Results (Data) (26 pts)1. Results
are clearly recorded and organized. 2. Figures are of publish
quality and clearly labeled and easy to follow with written results.
3. Easy for the reader to see trends in the results. 4. Clearly
transitioned and incorporated new data into results.One of the "excellent" items is
missing.Two of the "excellent" items are missing.Three of the "excellent" items are
missing.Discussion/Conclusion (28 pts)1. Summarizes essential data used to draw
conclusions. 2. Discusses application and
implications of results. ("real world connections"). 3. Puts findings
into context of previously published results. 4. Describes future
direction of project. 5. Concludes with an overall statement (e.g., In
summary, this work...). One of the "excellent" items is missing.Two of the "excellent"
items are missing.Three of the "excellent" items are missing.References (6 pts)Clearly
organized.Missing appropriate citations.Format and Grammar (6 pts) Neat organized with
clear headings, few grammatical and spelling errors.Somewhat lacking in organization,
multiple spelling and grammar errors, not neat.Comments:
Transgenes –definition, design and testing
A Typical Eukaryotic GeneThe typical eukaryotic gene is composed of a cis-regulatory
domain as well as the sequences required to encode the protein Start of
transcriptionExons/intronsCis-regulatory region
The cis-regulatory domain contains binding sites for transcription factors –the binding of
these proteins regulates gene expression
In multicellular organisms, genes can be expressed in different cell types (like the
Cionaembryos to the left –green color), at different times and at different levelsThe cis-

15. regulatory sequences of the gene regulate temporal, spatial and quantitative expression
A transgene combines these cis-regulatory sequences with a reporter gene (like GFP). When
expressed in the cell/tissue/organism it should reflect the expression of the endogenous
gene.To know if your transgene works correctly, you need to have characterize the
expression of the endogenous gene you are interested in studying: i.e. its spatial, temporal
and quantitative expression. This can be analyzed using in situ hybridization (see next
slide).You then generate a transgenic embryo and analyze the expression of the transgene.
If it matches the expression pattern of the endogenous gene, then you have likely captured
the relevant regulatory information on the piece of cis-regulatory sequence you used in
your transgene.
In situ hybridization can be used to localize gene expression in cells, tissues and
organismsThe purple stain shows you where the gene is expressed
Steps to design/test a transgene:•Start with genome sequence 5’ to the start of the gene of
interest –why? This is commonly where most regulatory sequence information
exists•Identify appropriate restriction enzymes to use to clone the DNA fragment•Create a
translation fusion between exon of the gene and the reporter protein –why? Because
studies have shown that including an intron in your transgene can enhance its expression.
There can also be regulatory elements located in introns•Express your transgene in your
organism. If it is expressed the same as the exogenous gene, you most likely have all of the
required regulatory elements contained within your fragment. If not, you are missing
important regulatory regions and must locate those. They could be in other introns,
downstream of the gene of further upstream. You would need to design and test additional
transgene reporters.Your goal for this exercise is to design a transgene reporter for the gene
your group has been working on.
Go to ensemble.org and find the Brachyury gene as you have done beforeHere we want to
grad sequence upstream (i.e. 5’) of the gene. The arrow in the name is pointing to the right
so we are in the correct orientation. Click and drag to zoom in a bit more.
Brachyury geneUpstream geneWe want sequence from this region, type the values above to
zoom inType in range 3900-6600
Click on export data to get a fasta-formatted sequence of this region.Note, if your gene is
oriented in the opposite direction, you can still follow these instructions to get your
sequence. In Snapgeneyou can easily reverse your sequence to work with it.
Hit next, then select “text” that will give you the sequence you see to the right. Grab that
sequence…
Using Snapgene, add this sequence as a new sequencePut the sequence in “sequence view”,
we are going to annotate the first couple of coding exons to help us design our transgene.
In ensembl, pull up the Brachyury exon sequencesWe just need to first two exons, copy the
first exon sequence (just the blue sequence is ok)
In Snapgene, navigate to align 2 sequences
The Brachyury sequence you grabbedThe first exon sequence you just copiedAlign them!
Note the endpoints, then let’s annotate the sequence
Select the same region of sequence (2394-2462) then click on add features-> add feature

16. Name itExon or cds, once you hit translate it will force it to be cdsClick on feature
translation options to select the proper reading frame. This is correct –hint you get compare
to the Brachyury protein sequence to confirm.
Now do the same steps from exon 2Now both exons are annotated and you have the correct
reading frames aligned with you sequence. This will help you with getting the restriction
enzyme site in the proper reading frame.
This is the transgene cloning vectorIt has the following parts: AmpR(selectable marker)Ori
–plasmid origin of replicationMCS –multiple cloning siteCrHistone–histone coding
regionYFP –YFP coding regionCrBetaActin3’ UTR –3’UTRYou will be cloning your genome
fragment into the MCS and cloning it in-frame with the histone coding region. This will
produce a reporter transgene that will express YFP in the nuclei of cells (because YFP is
fused to a histone)
Go to sequence viewNow you can see the enzymes in the polylinkerand the reading frame
relative to the histone sequence (blue box).Now you want to go the Brachyury sequence in
Snapgeneand see if it has any of the polylinkersequences. If it does, skip those and make
note of which polylinkerenzymes you could use to clone your transgene. Note that both
PvuIIand BamHIare not in bold, indicating these are not unique sites –don’t use them!To
save you some time, you can go to enzymes-> noncuttersand see a list of enzymes that don’t
cut your sequence (you can export this information to make it easier to search)
These PL enzymes do not cut and are OK to
use:AatIINotIXhoIHindIIISphIPstISalISmaIAcc65iThese enzymes that you can use are listed
from the 5’ to the 3’ of the MCSIf you look at the MCS, you’ll see a stop codon present 5’ of
the HindIIIsite. Therefore, make sure your 3’ most enzyme is between HindIIIand Acc65i.
Let’s use XhoIfor the 5’ end of DNA fragment and Acc65i for the 3’ end of the fragment
Start with the 5’ (easy) endGrab the first 25-30 nucleotides and check against the NEB Q5
DNA polymerase calculator from last labhttps://tmcalculator.neb.com/#!/mainNow add a
few ntplus your RE site for XhoI:5’ (forward) primer:
gcgctcgagCTTCTCATAGAAATACAATACAAGTTACG
The 3’ primer is a bit more complex….You can hover the mouse over the RE site to
determine the sequence and see how it lines up with the coding region.For Acc65i, the site
is GGATCC and this is the reading frame that we to maintain:cgGGTA CcaR V PWhat we
want to do in this case is to find a region in exon 2, find a codon with “G” in position 3 and
then add GTACCgcgto add our RE
Here we use this methionineGATCCAACTGCGATGTATTCCGTCATG gtaccgcg
Add your primers to the Brachyury sequence
Add your primers to the Brachyury sequenceDon’t forget to reverse compliment the reverse
primer!
Now let’s build the reporterUse your primers to generate a PCR fragment of the genomic
region you want to cloneUsing this fragment, subcloneit into the transgene vector with
XhoIand Acc65iUse the same steps you did previously
The resulting clone, check the sequence view.
Here is where your 3’ end of your clone went into the vectorNotice that the reading runs
from exon 2 into the histone sequence without frameshifts

17. The sequence you just cloned corresponds the region known to be an active regulatory
domain for the CionaBrachyury gene.If you put this transgene into the ascidian Ciona, the
cells where Brachyury is expressed (the notochord cells) would be expressing the transgene
as shown in an image of living embryos above.
AssignmentUsing the Cionagene assigned to your group from last week, build a transgene
following the example in these slidesDocument your steps, screen grabs can be used if
needed:1)What region did you select (like slide #9).2)Show your sequence annotation (like
slide #20). If your gene only has one exon, just use that single exon3)Indicate which
enzymes you can use (like slide 23). If you can’t identify a pair of enzymes, what could you
do?4)Show your forward and reverse primers (like slides 24 and 26)5)Show your
completed transgene (slide 30)6)Show a closeupof the 3’ cloning region indicating that you
cloned your sequence in-frame with the reporter construct (like slide 31).
The Polymerase Chain Reaction (PCR) and PCR-based cloning•PCR has revolutionized the
way in which nucleic acids can be identified, amplified and used in molecular biology
Key milestones in the development of PCRhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcrWatson and CrickNobel Prize
for DNA structureArthur and SylvyKornbergNobel Prize for DNA replicationHar
KhoranaNobel Prize for genetic codeKleppe, a postdoc of Khorana’s proposed 2 primer
system but never did the experiment!Kary MullisBrock -1969 isolated a thermophile from
Yellowstone NP -Thermus aquaticus. 1976 -Chien and Trelapurified DNA polymerase
showing it was active at 80C, clone reported in 1988Needed to add enzymes each cycle,
needed to move tube by hand to different water baths
•This video provides a brief review of the PCR process:
https://www.youtube.com/watch?v=c07_5BfIDTw
The implementation of PCR required several key developments•Discovery and
characteristic of DNA polymerase enzymes•Generation of oligonucleotides which could be
used as “primers”•Realization that orienting primers opposed to one another could cause
exponential amplification•Isolation and cloning of thermostable DNA polymerase (Taq
polymerase)•Engineering of thermocycler machines that could automatically change
sample temperatures during the process.
Key steps in the PCR cycle: Denaturation, Annealing, Extensionhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcr
Primer design•Although designing primers seems straightforward, there are a variety of
issues that one must think about when planning for an experiment. The following video
outlines some of these issues which we will
discuss.•https://www.youtube.com/watch?v=mcOwlFVEino
Primer design considerations –length, GC content and TmGC content, length and melting
temperature. These are related as follows: longer primers and/or those with higher GC
content have higher melting points. Generally, you want to end the 3’ primer sequence with
a “C” or “G” (what is known as a “GC” clamp). This may require you to move the location of
the primer along your sequence to be amplified. Primers should have a Tm within about 5C
of each other and within the range of 65-75C, this will help to avoid mis-
primingOligonucleotides are synthesized 3’ to 5’ and the longer the primer, the lower the

18. percentage of full-length product in the synthesis reaction% Full length product =
(efficiency)(n-1) where efficiency is the synthesis coupling efficiency (typically about 99%)
and n is the number of cycles. For a 50 ntlong primer:% full product = (0.99)49= 61%. 49
cycles are needed to make a 50-mer
Primer design considerations –sequence composition•Avoid runs of 4 or more bases
(“AAAA” or “CCCC”)•Avoid dinucleotide repeats (i.e. “ATATATAT”)•Avoid intra-or inter-
primer homology as this will cause the primers to bind internally or with each other
interfering with the amplification of your product.Hairpin from internal bindingInter-
primer homologyLastly, if you plan to incorporate restriction enzymes sites for cloning, add
those with 3-5 bp additional sequence to allow efficient enzyme activity
Given the previous constraints, software packages have been developed to assist with
primer designIn this exercise we will be amplifying specific regions of genes and this
typically requires one to hand-design primers sequences. We will use a tool called
PrimerBlastto help us determine what the best design would be.First we will design primer
sets to specifically amplify the coding region of a gene so that it can be cloned into an
expression vector with a specific reading frame.Second, we will work to design primers to
make a transgene reporter.The following slides will walk you through the process.
Sequences to useFor simplicity, you will be working with several different sequences from
the ascidian Cionaintestinalis. Recall that you already have worked with Cionasequences at
ensemble, here you will be provided with community-annotated transcripts that include 5’
and 3’ UTRs.The cloning vectors will also be explained and annotatedFirst, let’s translate the
BrachyurycDNA sequence into the correct reading frame.
Use https://web.expasy.org/translate/to translate Make sure you select:the bottom radio
buttonForward strandThen hit translate
Scrolling down shows frame 2 as the correct reading
framecacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgt
catcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTP
PSLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonHINT –put
your sequences in font Courier New so everything aligns
We want to design PCR primers that will amplify the entire open reading frame from the
start to the stop
codoncacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacg
tcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonLet’s make a first pass at making a primer. Grab about 7-8 codons of sequence,
stopping at position 2 of the last codon. Why should you stop there?
Check your primer sequence for use with Q5 DNA polymerase (a common PCR enzyme)
https://tmcalculator.neb.com/#!/mainLooks good, now do the second primer
atgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSLtga
cgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonThese look good, let’s
check them for other characteristics
Go to Primer Blast: https://www.ncbi.nlm.nih.gov/tools/primer-blast/BrachyurycDNA
sequenceForward primerReverse primerFor your reverse primer use this reverse-

19. compliment tool: https://www.bioinformatics.org/sms2/rev_comp.htmlUse these values
Scroll down, remove humans and add CionaintestinalisThis will allow the software to check
your primer design against the genome to help identify off-target hitsHit “get primers” and
wait for search to complete.
Output –not bad, but some 3’ and self complimentarityForward: atgttttcgaagctcagtgcgag3’
self comp, can form hairpin in primerPoly T run, sometimes you can’t avoidClick here for
detailed results
Tweak the forward
primercacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgac
gtcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonNew forward: atgttttcgaagctcagtgc. This is shorter so the Tm decreases, make the
reverse primer shorter
tooatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSL
tgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtga
Primers look good, let’s do a virtual PCR
PCR test:
https://www.bioinformatics.org/sms2/pcr_products.htmlBrachyurysequenceForward and
reverse primers (make sure to reverse compliment the reverse primer)
PCR Products results –looks like the correctregion>1359 bpproduct from linear template
KH.S1404.1.v1.A.nonSL1-1 Brachyury, base 26 to base 1384 (Forward -Reverse).
ATGTTTTCGAAGCTCAGTGCGAGTTACAAACCTATAATGACGTCATCAGATAGTAAGTTA
GCAGGTATGACGTCATCAGAATCAATTGAAACATGTGCAGTAAAGATGGCGCTAATAGAG
CATGGTTTATGGTCGAGGTTTCACGCGTTTGTCAACGAGATGATTGTGACAAAAAATGGA
CGACGAATGTTTCCGGTTCTTAAAACATCGATTACGGGACTTGATCCAACTGCGATGTAT
TCCGTCATGCTTGACTTTGTACCCGTGGATAACAATAGATGGAAATATGTGAACGGTGAG
TGGATCCCCGGGGGAAAACCCGAACCCCATGTTTCGTCATGTGCTTATATTCACCCCGAT
TCACCCAACTTTGGTTCGCACTGGATGAAACAACCCGTTGGTTTCAGTCGCGTCAAACTT
ACTAATAAAGCTACAGGGAACCCCCAGCAAATAATGTTGAACTCTCTGCATAAATACGAA
CCCAGGATCCACATCATGCGCGTTGGGGGCGCTGAATCGCAACAAGTCGTTGCCTCGCAT
TCATTTCAGGAGACAAGGTTCATTGCTGTTACTGCTTACCAAAACGAAGATGTTACTTCG
CTCAAGATAAAATATAATCCTTTCGCCAAAGCTTTCCTCGACGCAAAGGAGAGCCGCTCA
GGGAGTGAAAATTATTTTAAAGATTCAACGAAAGCGGGTTCGTCGCAAAATTATTCGAGA
GCGAACACATGGACGGCAAATCAAAGCAACCCAACATTCAATCAATGTCAGTATGAATCA
GGGATTCCCCCATTCCATTACCCCATCCCAAACCAACCGAAAACAACAAGAGAGCGGCGC
ATGAGCCGCACACAAAGAAGCCATCCCTACAAACCATCCACAACGCAAATTTATCAAGAT
TTCCAACCAACCAATTACCCCGCCCTACCAAACGACCAATGGCAATCAAGTATCGAAGGT
CATGAACTCGATGAGGGACACTTTAGTCTCGAACCAGTCTCTGTAGATGACGTCACCGCC
TTAGGATTCGACACCCCGCATCAGGGTTTTGCAACAAACGATCTTCTTTCAATAGAGCCT
TCCTATTCGTTAGATTATCCTCAATTCACGACCACGTGGTCCTACCCGACCAATCACATG
CCAGGATATGTTTCAAACAGTCCAATCAGAAGCCTGGAACAACCGGAGTACTTTTATAGA
GGCTATGACGTATCACAGCAAAATTACGTCACGATGACGTCAGCAGATGTTAGTAATGTG
ACGTCATCATTATACGAAACTCCGTCACCGGGGGTCATGCAAAGGCCACAAGAAGATTTT

20. TCAATCGCGTATACGCCGCTTACGCCACCTTCTTTGTGACheck the ORF at
https://web.expasy.org/translate/
Looks good!
Now let’s clone this sequence into a bacterial expression vector (plasmid) so we can make
BrachyuryproteinReview that plasmids are circular DNA pieces present in bacteria.
Researchers have adapted these molecules for use in molecular BiologyIf you’d like a
reminder on basic cloning approaches, please review these videos:Restriction enzyme
cloning: https://www.neb.com/tools-and-resources/video-library/cloning-with-
restriction-enzymesOur approach: https://www.neb.com/tools-and-resources/video-
library/overview-of-traditional-cloning
Bacterial vector for making proteinsFree viewer at snapgene.com. This will let you do basic
analysis like see where restriction enzymes cutVector = pTrcHisA, B or C
The polylinker(cloning site) has recognition sites for different restriction enzymesWe need
to identify the correct enzymes to use and add those to our PCR primers.
This vector has three versions which changes the reading frame around the restriction
enzyme sitesABCSince we can use PCR to set the reading frame, we can just focus on one
vector, so we will use pTrcHisC
Here reading frame is importantBamHITGGATCCGACCTAGGCW
IBglIIGAGATCTGCTCTATGACD LAcc65i/KpnIGGTACCTAACCCTGGTV PRE sites are in
PURPLECodon is in REDTo express our protein, we need to add the correct RE sites to our
primers with the correct reading frameThe enzymes in our polylinkerare: BamHI, BglII, PstI,
Acc65i, EcoRIand HindIIIOur DNA insert must be oriented ATG -> stop in this direction
Let’s see what enzymes we can use: https://nc3.neb.com/NEBcutter/Paste your FASTA
sequence in, preferably from your PCR productOverviewExamine enzyme listLook at zero
cutters –these are enzymes that don’t cut our PCR product
BamHIcuts our DNABglIIdoes not cutPstIdoes not cutAcc65i does not cutEcoRIdoes not
cutHindIIIcuts our DNALet’s use Acc65i and EcoRIThese let us insert our DNA in the proper
orientationThe enzymes in our polylinkerare: BamHI, BglII, PstI, Acc65i, EcoRIand
HindIIIWhat happens if we can’t usable RE sites??
We need to worry about the reading frame for Acc65i on our clone. Because EcoRIis after
the stop codon, reading frame doesn’t matterAcc65i/KpnIGGTACCTAACCCTGGTV
PcacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgtcatc
agatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonOur primer needs to preserve this reading frame: G GTA CCT AOur primer sequence
starts with the start codon ATGIf we keep the prolinecodon from the RE and simply add our
primer sequence we are good to go:G GTA CCT atgttttcgaagctcagtgcgV P M F S K L S
ALastly, add 3 nucleotides to the 5Õ end of the primer so the enzyme can cut: CgcG GTA CCT
atgttttcgaagctcagtgcg
The reverse primer is
easieratgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPP
SLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtgaCttacgccaccttctttgtgaGAATTCgcgEcoRIRemember to reverse
compliment:cgcGAATTCtcacaaagaaggtggcgtaaG

21. Group assignmentEach group will be given a gene to work with, these are provided
separately. I’ve also provided the Brachyurysequence used in the powerpointwalk-through.
1.For your gene, first identify the ORF as on slide 12. Show the correct start and stop
codons2.Design your first round of primers like slides 13-153.Check your primers in Primer
Blast and report the output (slide 18). If you find there are additional matches, you can
always blast your ntsequence at ncbito identify if you are hitting alternative splice forms of
your gene. If you do, will this interfere with your PCR amplification? Why or why not?
4.Tweak primers if needed and report like slides 19-205.Check your PCR product like on
slides 22 and 236.Check your sequence (the PCR product) on NEBCutterand report the
results (i.e. slides 29/30). Which enzymes can you use for your primers. If you can’t use any
enzymes, what could you do? Hint: sequence mutation would be needed in your PCR
product –what would you need to do?7.Report final forward and reverse primer design
(like slides 31-32)
Website referencesORF finder: https://web.expasy.org/translate/Primer Blast:
https://www.ncbi.nlm.nih.gov/tools/primer-blast/Reverse compliment:
https://www.bioinformatics.org/sms2/rev_comp.htmlNEB Q5 calculator:
https://tmcalculator.neb.com/#!/mainNEB Cutter: https://nc3.neb.com/NEBcutterPCR
simulation: https://www.bioinformatics.org/sms2/pcr_products.html
Gene orthologues, genome browsers and gene landmarksIn this week’s lab we will build
upon the exercises from last week (extracting sequences from DBs, BLAST searching,
translating)Here we use a test gene called Brachyury to explore genome browsers and gene
structure These exercises will prepare you for the next sequence exercises –designing
primers to clone and mutate genes and build transgene reporters.
Important Links•Ensembl genome website:
https://uswest.ensembl.org/index.html•Ensembl metazoa:
https://metazoa.ensembl.org/index.html•https://www.youtube.com/watch?v=C2g37X_uM
ok•The above video link provides a walk through on how to browse genomes and genes at
ensembl
Brachyury is a transcription factor important for early development –it will be our example
proteinhttps://en.wikipedia.org/wiki/T-box_transcription_factor_TThis is a model of the
Brachyury protein binding to DNA (purple molecule)
Brachyury belongs to a family of genes called Tbox genes.
https://www.pnas.org/doi/10.1073/pnas.1309748110Brachyury itself is widely
distributed among metazoans and even fungiWe will use this characteristic to learn how to
explore orthologues of Brachyury in different animal genomes.
Select animal genomes –based on a sample of Biology faculty model
organismsDrosophila(fruit fly): Bernstein, Cripps, Capelson
https://uswest.ensembl.org/Drosophila_melanogaster/Info/IndexSea Urchin:
SchrankelPlanarian: ZayasAscidian:
Zellerhttps://metazoa.ensembl.org/Lytechinus_variegatus_gca018143015v1/Info/Indexhtt
ps://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Indexhttps://usw
est.ensembl.org/Ciona_intestinalis/Info/IndexAll of these animals have much smaller
genomes than vertebrates (mice, humans, fish) making some of our exercises easier to do. C.

22. elegans: Luallenhttps://uswest.ensembl.org/Caenorhabditis_elegans/Info/Index
Let’s get started! Go to ensemble.orgEnsembl maintains genome assemblies for dozens of
organisms and is a one-stop place for doing comparative genome browsing. Your TAs may
use other genome sites for their own research –ask them about it!
Retrieve the brachyury protein from the mouse genome:Select the mouse genomeType
“brachyury” in the box and hit go
Find the circled link and click it
General information, note the accession number (ENSMU…)Position in genome,
chromosome 17,Nucleotides 8653255 to 8661328Forward strandLinks to similar proteins
Mouse Brachyury on the genome browserA genome browser is typically web-based and
shows a variety of information that you can explore to learn about genes and
genomesBefore moving on, let’s explore the browser interface and the information it
displays.Click here!
Mouse Brachyury on the genome browserClick here!
Detailed viewDirection of transcription ->8 exons, 7 introns1234 56785’UTR3’UTRThe rest
of the exons have coding sequenceClick on cDNA after viewing slide
cDNA sequenceNote the sequence is colored coded and the translation is also shown
Exon view. Note the exon sequence is shown separately from the intron sequences. The UTR
sequences are also color coded
Protein view.You can download the Fasta formatted protein sequence
Mouse Brachyury on the genome browserGo back to this viewClick on “region in detail”
Chromosome and positionChromosomal region surrounding the Brachyury gene, ~500
MBZoom controls for belowBrachyury gene
Arrows shift view upstream or downstreamThese are called “tracks” and describe various
kinds of data to display such as predicted/known genes, SNPs, regulatory gene information,
etcT-201 is BrachyurypromoterTry zooming outNote that there are several alternative
splice forms of Brachyury
BrachyurypromoterenhancerIf you wanted to build a transgene, you would need to include
promoter/enhancer information
Go back to this page, click on tab labeled Gene: TIf you click on orthologues, you can get
other proteins that match, but we’ll do a Blast instead
Paste in your mouse Fasta file for Brachyury, select Drosophila, C. elegansand Ciona
intestinalis for other species and then run Blast
Paste in your mouse Fasta file for Brachyury, select Drosophilaand Ciona interstinalis for
other species and then run BlastSearch the protein DBs for your species –why? This will
allow you to BLAST against a protein database that will then allow you to find the
corresponding genomic region. BLASTing against the genome will split your hits up among
the various exons of the gene.
Check your results!
For Ciona, the top hit is the CionaBrachyury geneClick on genomic location
Brachyury!You may need to adjust the view to see this image –zoom out move the window
An easy way to get the view you want is to zoom out, then drag a box about the region you
want to focus on. Select jump to region.

23. Now you have a centered view of the CionaBrachyury gene. Instead of zooming out and
selecting the region, you can just type in the coordinatesThis small > symbol tells you the
direction of transcription
Now look at the Drosophilahit repeating the steps needed to view the gene plotted on the
genome.This small > symbol tells you the direction of transcriptionNote: although the
Brachyury gene is found in a wide group of organisms, it has been lost in C. elegans and in
the Planarian Schmidtea mediterranea.
Compare the three Brachyury gene structuresmouseDrosophilaCionaHow many exons?
How many BP in each genome? Characterize the UTRs/coding regionsAlternative
transcripts
exonexonexonexonexonexonexonExon-intronboundaryHow to identify intron-exon
boundaries. Each line of nucleotides is the same length, so you can easily line them upThis is
the mouse Brachyury gene
Helpful hints:1)Paste your cDNA sequence into a word or google doc. Take a few minutes to
clean up the view so it is easy to read.2)Make sure you put the text into “courier new” font
and adjust the font size until it looks nice3)Now just search for the sequence from the exon
view in your document. The first exon boundary ends at “GAACGGCAG”4)You can use the
amino coordinates to help you find the junctions in your clustalw alignment (next slide)
T-201
MSSPGTESAGKSLQYRVDHLLSAVESELQAGSEKGDPTERELRVGLEESELWLRFKELTN60brach
yury-201 --------------------MTSSDSKLAGMTSSESIETCAVKMALIEHGLWSRFHAFVN40::: :*:*
. :.. . :::.* * ** **: :.*T-201
EMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVTADNHRWKYVNGEWVPGGKPEPQAP120
brachyury-201
EMIVTKNGRRMFPVLKTSITGLDPTAMYSVMLDFVPVDNNRWKYVNGEWIPGGKPEPHVS100**
**************..::****.****.:**** .**:*********:*******:. T-201
SCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGG-GQIMLNSLHKYEPRIHIVRVGGP-
178brachyury-201
SCAYIHPDSPNFGSHWMKQPVGFSRVKLTNKATGNPQQIMLNSLHKYEPRIHIMRVGGAE160**.*
*********:**** **.**:****** .*. ****************:**** T-201 -
QRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERNDHKDVMEEPGDCQ237brach
yury-201
SQQVVASHSFQETRFIAVTAYQNEDVTSLKIKYNPFAKAFLDAKERSGSENYFKDSTKAG220*::::**
.* **:**********::*:******************.. :: ::: .. T-201 Q-PGYSQ-WGWLVPGAG---
TLCPPASSHPQFGGSLSLPSTHGCER-YPALRNHRSSPYP291brachyury-201
SSQNYSRANTWTANQSNPTFNQCQYESGIPPFHYPI--PNQPKTTRERRMSRTQRSHPYK278. .**:
* . :. . * *. * * : *. * *.:** ** T-201 SPYAHRNSSPTYADNSSACLSMLQSHDNWSSLG-
----VPGHTSMLPVSHNASP------340brachyury-201 PSTT-----QIYQDFQPTNYP-
ALPNDQWQSSIEGHELDEGHFSLEPVSVDDVTALGFDT332: * * . : :*:*.* ** *: *** :
T-201 -----------------PTGSSQYPSLWSVSNGTITPGSQTAGVS--
NGLGAQFFRGSPA381brachyury-201 PHQGFATNDLLSIEPSYSLDYPQFTTTWSYPTNHMP-
GYVSNSPIRSLEQPEYFYRGYDV391. *: : ** .. : * : . *:** .T-201
HYTPLTHTVSAATSSSSGSPMYEGAATVTDISDSQYDTAQSLLIASWTPVSPPSM436brachyury-

24. 201 SQQNYVTMT-SADVSNVTSSLYETPS------PGVMQRPQEDFSIAYTPLTPPSL439. . :* *. *
:** : . : *. : ::**::***:Just the mouse (top) and Ciona (bottom) sequences are shown.Use
font “courier new” to get your alignment to look nice.In this example, the amino acids
located at the exon-intron borders have been highlightedNote that some boundaries are
conserved(i.e. same relative positions in proteins, red circles)While others are not (blue
circles)
Analysis and questions for the exampleCompare and contrast your three brachyury genes
(mouse, Ciona, Drosophila):Do the genes have the same numbers of exons in each
species?Do the genes span the same amount of space (length) in the genome?Are the 5’ and
3’ UTR regions the same length? Are the UTRs located on their own exon or on an exon
which contains coding region?Are the exons located at the same positions within each gene?
You will need to extract the exon sequence for each gene for this and compare to the cDNA
sequence for eachPerform a clustalw alignment of your 5 proteins and annotate the
positions of the intron/exon boundaries on the alignment (this is on slide 32 for you)
Data needed for assignments•Each group has a gene to analyze in 5 genomes (Drosophila,
Ciona, C. elegans, Sea Urchin and Planarian. These are listed below.•Use the accession
number to find the corresponding mouse protein sequence, use that in a blast search•For
Drosophila, C.elegans and Ciona intestinalis: https://uswest.ensembl.org/index.htmlFrom
that site, select blast and input your protein sequence. Be sure to select all three species so
that you only need to do one blast run for these 3 species•For the sea urchin, use this link:
https://metazoa.ensembl.org/Strongylocentrotus_purpuratus/Info/Indexand select blast
from the top menu•For planarian, use this link:
https://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Index/and
select blast from the top menu•Retrieve the protein sequence from each genome and do a
clustalw multiple alignment (we did that last week). This information will be used later as in
the example.•Put screen grabs of the genes for each species in your document (i.e. like on
slide 29)•Retrieve the exon and cDNA sequences for each gene, you will need that to answer
some questions.
If your gene has alternative splice forms, use the longest one with the most exons
Mouse gene assignments by
group1ENSMUSP000000253742ENSMUSP000000411183MGP_WSBEiJ_P00627004ENSMU
SP000000692775ENSMUSP000000060716ENSMUSP000001273967ENSMUSP000000580
208ENSMUSP00000044879
What to turn in:•Compare and contrast your five genes (C. elegans, sea urchin, planarian,
Ciona, Drosophila). Make a figure like on slide 29 and answer the next five questions•Do the
genes have the same numbers of exons in each species?•Do the genes span the same
amount of space (length) in the genome?•Are the 5’ and 3’ UTR regions the same length?
•Are the UTRs located on their own exon or on an exon which contains coding region?•Do
all of the genes have annotated 5’ and 3’ UTRs? If not, why do you think that is?•Perform a
clustalw alignment of your 5 proteins and annotate the positions of the intron/exon
boundaries on the alignment like on slide 32.•Are the exons located at the same positions
within each gene? Why do you think some boundaries are preserved while others are
unique?

25. Biology 366L 2023Week of January 30th
A brief review •The central dogma of molecular biology•Gene structure•Location of genes
in chromosomes
The central dogma of molecular biology:DNA -> RNA ->
Proteinhttps://www.youtube.com/watch?v=gG7uCskUOrAIn general, information is stored
in the genome as DNAis Transcribedinto RNA and then Translatedinto Protein.
The central dogma of molecular
biologyhttps://www.britannica.com/science/geneInformation flow:DNA -> RNA ->
ProteinIntrons spliced outA more detailed review can be found here:
https://openlab.citytech.cuny.edu/openstax-bio/exam-4/gene-function/
The coding region encodes the amino acids for the corresponding
proteinhttps://www.researchgate.net/figure/The-structure-of-a-gene-and-the-control-of-
gene-expression-in-eukaryotes-Initially-RNA_fig2_263714372Open reading frame
Genes are located along chromosomes within a eukaryotic
organismhttps://sitn.hms.harvard.edu/flash/2012/issue127a/
Our genetic information is stored in our chromosomesGiven either a nucleic acid or protein
sequence, researchers can use various bioinformatic approaches to identify and clone a
gene, build transgene reports, and manipulate gene functionHere we will introduce a
variety of algorithms and databases that are used for such purposes.
Sequence search algorithmsProbably one of the most-utilized algorithms for sequence
searching is BLASTBLAST stands for Basic Local Alignment Search ToolBLAST website:
https://blast.ncbi.nlm.nih.gov/Blast.cgi
There are several “flavors” of BLAST, depending on whether you are searching with nucleic
acid or protein sequences•BlastN –nucleotide vs nucleotide•BlastP –protein vs
protein•BlastX –nucleotide vs protein•tblastN –protein vs translated nucleotide•The above
indicate query (the sequence you have) vs subject (the DB)
You want to start with a FASTA formatted sequence fileFasta files begin with a “>”
character, then a description. The sequence (either nucleotide or protein) follows on
subsequent
lines>avGFPMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW
PTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR
IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIG
DGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKYou can retrieve
sequences from genbank, to get the GFP sequence above, retrieve the sequence using the
sequence identifier “AAA27722.1” This is also referred to as an accession number.
This link will give you the corresponding nucleotide sequence
Fasta formatted sequence
Use SMS to translate a sequenceUse this accession: L29345.1To retrieve the GFP mRNA
sequenceNote: although there are many websites that can perform sequence manipulations,
etc., many labs have their own local software for performing these functions.Which reading
frame do you need to translate the mRNA into to obtain the GFP protein sequence?
Example BlastP runProtein query sequence in Fasta formatDefault DB is non-redundant

26. proteinsBlastP is selected
BLAST output is scored so you can analyze the resulting “hits” Evalue: smaller = better
hitQuery cover: how much of your query sequence was matchedPer identityAccession: link
to “hit” sequence
Clicking on graphical summary gives you a visual representation of hitsClick on conserved
domains to learn about detected protein domains
GFP has a single, conserved domain
The alignments tab shows you the aligned query and subject sequences
Fluorescent proteins are used for many different research purposesVideo link about GFP
(can show in lab): https://www.youtube.com/watch?v=Z4vJ8rQCNggVideo link for more
GFP information (optional, don’t show in lab):
https://www.youtube.com/watch?v=qK9aYnkIr3w
FPBase –a curated database for fluorescent proteinsExplore if interested
Protein structure -alphafoldAlphafold –computational protein foldingYou can learn more
about alphafold here:https://www.deepmind.com/research/highlighted-
research/alphafoldInput "green fluorescent protein" Aequorea into the search box to
explore
With AlphaFold you can interact with the predicted protein structure
Sequence multiple alignmentsYou may wish to determine how similar different sequences
are to one another, to do that you can use a tool such as ClustaW:
https://www.ebi.ac.uk/Tools/msa/clustalo/Here you can provide multiple sequences to
determine similarities across the sequence. The corresponding document in Canvas has
more specific instructions.
Output codes:* = exact match: = strong conservation. = weak conservation= mismatch
UniProt is a protein repository
Expasy is a portal to access various suites of bioinformatic tools
AssignmentYour instructorwill organize you into working groups. Each group will be
provided an accession # for a gene of interest. You can make a simple group report using
google sheets or the office suite. Use text, screen grabs and pictures in your findings.Given
this accession # please do the following:1)Retrieve the nucleotide and protein sequences,
what species is you sequence from?2)Given the gene name from the sequence file, use
resources at NCBI to determine the function of your gene3)Using the mRNA sequence for
your gene, use SMS to translate this sequence into the correct protein –which reading frame
was needed?4)Perform a Blast search using the nucleotide sequence and then the protein
sequence, report on the top 5 hits. Do they correspond to the same gene in each search?
Why or why not?5)From your protein blast search, identify conserved domains (if any).
What are they and what is their function?6)With the top 5 proteins from your blast search,
perform a multiple alignment using ClustalW. Are these proteins similar? Does the region of
similarity overlap with the regions of conserved domains? Why or why not?7)With your
protein sequence for your gene, locate its predicted structure on AlphaFold–add a screen
grab to your report.