pre-lab information about the restriction digest and bacterial transformation labs for ap biology

1. Hello you.Try this.Hello you.Try this.
2-1-17
If an enzyme was
able to cut this
DNA at each “x”,
how many
fragments of DNA
would be
produced? What
fragment would be
largest? Smallest?
x
x x x
x

3. Your goal – to get theget the
basic idea of each labbasic idea of each lab –
what we will do for each
Today’s Goal

4. AP BiologyAP Biology
1.1.Restriction Digest and Analysis ofRestriction Digest and Analysis of
λ DNAλ DNA
2.2. BacterialTransformationBacterialTransformation
3.3.DNA Fingerprinting/Crime SceneDNA Fingerprinting/Crime Scene
AnalysisAnalysis

5. 1. Restriction Digest of Lambda DNA
1. Cut the DNA from a virus with enzymes and analyze
the results
2. BacterialTransformation
1. Cut out a gene from the DNA of one organism and
insert it into the DNA of another
3. DNA fingerprinting
1. Cut several DNA samples with enzymes and analyze
them to look for similarities & differences
Our 3 labs

6. Restriction Digest &
Analysis of Lambda DNA

7. Examine restriction enzymes and their use asExamine restriction enzymes and their use as
biotech toolsbiotech tools
Goal of this lab

8. useuse restriction enzymes
EcoR1, Pst1, & HindIIIEcoR1, Pst1, & HindIII to digest
(cut) bacteriophage lambda
DNA
 use gel electrophoresis to
examine these fragments of
DNA
What we will doWhat we will do

9. These enzymes cut DNA at very specific locationsThese enzymes cut DNA at very specific locations
Bacteria contain these enzymes as a defenseBacteria contain these enzymes as a defense
mechanism against phagesmechanism against phages
Restriction enzymes are named after the bacteria
from which they were first isolated
• EcoRI from Escherichia coli, strain RI
• HinDIII from Haemophilus influenzae, strain DIII
• PstI from Providencia stuartii, stain I
What are restriction enzymesrestriction enzymes?

10. • The DNA of a bacteriophage
• ~ 48,000 base pairs long
• 3 different enzymes to cut this DNA resulting in
different length restriction fragments
•one sample of DNA uncut for comparison
•One “ladder” or “marker” used for estimating sizes
of restriction fragments using gel electrophoresis
Lambda DNALambda DNA
(λ)(λ)

13. If a linear strand of
DNA is cut 4 times,
how many bands will
there be in our gel?
Restriction Enzymes

14. Electrophoresis - to carry w/ electricityElectrophoresis - to carry w/ electricity
Separates DNA fragments by sizeSeparates DNA fragments by size
DNA fragments loaded into an agarose gel slab
• Slab placed in chamber w/ conductive buffer
• Direct current passed through
• Since DNA molecules are negatively charged, they areSince DNA molecules are negatively charged, they are
drawn toward the positive pole (anode) when placed in andrawn toward the positive pole (anode) when placed in an
electric fieldelectric field
The gel acts like a sieveThe gel acts like a sieve through which smaller
fragments move more easily than larger ones
Over time, smaller fragments will travel farther than
larger ones
Agarose Gel Electrophoresis

15. Gel Electrophoresis
http://learn.genetics.utah.edu/c

16. DNA is colorless, so a loading dye is added toDNA is colorless, so a loading dye is added to
the DNA solutionthe DNA solution
•Does not stain the DNA
•Makes it easier to load the gels and monitor the
progress of the process

17. Each restriction enzyme producesEach restriction enzyme produces
unique banding patternsunique banding patterns
The relative size of the fragments cansize of the fragments can
be determinedbe determined by measuring how far
each band traveled from its origin

18. What are we doing overall in this
lab?
So…

19. pGLO BacterialTransformation Lab
Bacterial
Transformation

20. DNA splicing, or gene splicing, is the cutting and linking ofDNA splicing, or gene splicing, is the cutting and linking of
DNA molecules and it is one of the basic tools of modernDNA molecules and it is one of the basic tools of modern
biotechnologybiotechnology
The basic concept is to remove a functional DNA fragment,
like a gene, from one organism and combine it with the DNA
of another organism in order to study how the gene works
• The desired result is for the recipient organism to express the newlyThe desired result is for the recipient organism to express the newly
acquired geneacquired gene
Background

21. The ability to cut & paste, or cleave &
ligate, a functional piece of DNA is
what enables scientists to recombine
DNA molecules
First step is to locate the gene
Next use a restriction enzyme to cutuse a restriction enzyme to cut
out the gene from the rest of theout the gene from the rest of the
chromosomechromosome
Use the same enzyme to cut open thesame enzyme to cut open the
DNA of the recipient DNA and thenDNA of the recipient DNA and then
insert the fragmentinsert the fragment
Recombinant DNA technology

23. In this lab we will
perform genetic
transformation
•Genetic transformation
occurs when a cell takes
up and expresses a new
gene
We will learn about
moving DNA from one
organism to another
using a plasmidplasmid
Overview
GFP
Beta-lactamase
Ampicillin
Resistance

24. pGLO™
&
GFP
We will attempt to have our
bacteria express the gene for
green florescent protein
(GFP)

25. GFP is a visual markervisual marker
Study of biological processes
(example: synthesis of proteins)
Localization and regulation of gene
expression
Cell movement
Cell fate during development
Formation of different organs
Screenable marker to identify
transgenic organisms
Links toLinks to
Real-worldReal-world

26. In addition to their single circular
chromosome, bacteria often have 1 orbacteria often have 1 or
more small, circular pieces of DNA calledmore small, circular pieces of DNA called
plasmidsplasmids
5- 6 genes in a circle
Plasmids often contain genes that help
bacteria survive
Bacteria can share these plasmids with
each other
• Antibiotic resistance among bacteria is due to
plasmid sharing
What is a plasmidWhat is a plasmid?
Transmission
electron
micrograph

27. Contains theContains the genes for GFPgenes for GFP and the gene forand the gene for
ampicillin (antibiotic) resistanceampicillin (antibiotic) resistance
• It is called the pGLO plasmid
Our plasmid also has an operon that allows the gene
for GFP to be turned on and off
the sugar arabinose turns on the operon
Our plasmidOur plasmid

28. Beta Lactamase
•Ampicillin resistance
Green Fluorescent
Protein (GFP)
•Aequorea victoria
jellyfish gene
araC regulator protein
•Regulates GFP
transcription
operon
Green
fluorescent
protein
Gene to
break
down
antibiotic
Origin of
replication

29. BacterialTransformation
Beta lactamase
(ampicillin resistance)
pGLO plasmids
Bacterial
chromosomal
DNA
Cell wall
GFP

30. Transformation Procedure

31. What are we doing overall in this
lab?
So…

32. DNA
fingerprinting

33. • Crime scene
• Human relatedness
• Paternity
• Animal relatedness
Anthropology studies
Disease-causing organisms
Food identification
Human remains
Monitoring transplants
DNA
Fingerprinting
RealWorld
Applications

34. DNA Restriction Enzymes
• Evolved byEvolved by
bacteria tobacteria to
protect againstprotect against
viral DNAviral DNA
infectioninfection
•Endonucleases
= cleave within
DNA strands
•Over 3,000
known enzymes

35. No 2 people are exactly the
same genetically except…
• Identical siblings
All people share 99.9% same
DNA
• It is the 1/10 of 1% that makes us
different from each other
Scientists know where these
places are, and look there to
determine a DNA profile
DNA is unique

36. ~98% of the DNA in a human cell does not~98% of the DNA in a human cell does not
code for any proteincode for any protein
Some of this noncoding DNA is tandemtandem
repeats:repeats:
•The same DNA pattern repeated over and over
•For example: ACACACACAC –or-
GTCGTCGTCGTC
Noncoding DNA

37. How many repeats there are in the DNA varies fromHow many repeats there are in the DNA varies from
person to personperson to person
For example, suppose chromosome 17 had a tandem
repeat of ATCGATCGATCG
• Some people would have 3 repeats of that sequence
• Some people would have 4 repeats of that sequence
• Some people would have 11 repeats of that sequence, and so
on
Noncoding DNA

38. Scientists use the tandem repeats in a person toScientists use the tandem repeats in a person to
create a DNA profile or “fingerprint”create a DNA profile or “fingerprint”
Noncoding DNA

39. Step 1:
•Get a DNA sample (make copies of it in a
lab)
Create a DNA profile (DNA
fingerprint)

40. Step 2: cut the DNAcut the DNA usingusing
restriction enzymesrestriction enzymes
Create a DNA profile (DNA fingerprint)

42. Step 3: sort the DNA fragmentssort the DNA fragments by size using gelby size using gel
electrophoresiselectrophoresis
1. DNA is cut with restriction enzymes
2. The cut DNA is placed on a gel
3. Electric current runs through the gel
4. DNA is negative and moves toward the positive
charge (opposites attract)
5. The pieces of DNA separate by size; smallest move
farthest
Create a DNA profile (DNA fingerprint)

45. Step 4: compare the DNA to the
“Suspect” DNA
• Scientists typically look at 13 differentScientists typically look at 13 different
tandem repeat areas (loci)tandem repeat areas (loci)
•Looking at only 1 does not tell us very
much; many people could have that
one repeat
•Looking at 13, and being a match for
all 13, tells us a lot
• Being a perfect match for all 13 has aBeing a perfect match for all 13 has a
chance of 1 in 100 billion (there are 7chance of 1 in 100 billion (there are 7
billion people on the planet)billion people on the planet)
Create a DNA profile (DNA fingerprint)

46. DNA fingerprinting lab
Victim – Asuzena
S1:Yulissa
S2: Erica
S3: Rajvir
S4: Harinder
S5: Hernan

50. Determine
restriction fragment
sizes
•Create standard curve
using DNA marker
•Measure distance
traveled by restriction
fragments
•Determine size of
DNA fragments
Identify the related
samples
Analysis of Stained Gel

51. Electrical current carriescarries
negatively-charged DNAnegatively-charged DNA
through gel towardsthrough gel towards
positive (red) electrodepositive (red) electrode
Agarose Electrophoresis Loading
Power Supply
Buffer
Dyes
Agarose gel

52. • Agarose gel sieves DNADNA
fragments accordingfragments according
to sizeto size
– Small fragmentsSmall fragments
move farther thanmove farther than
large fragmentslarge fragments
Agarose Electrophoresis RunningElectrophoresis Running
Power Supply
Gel running

53. What are we doing overall in this
lab?
So…