This document discusses genomic and cDNA libraries. Genomic libraries are made from genomic DNA and represent all genes in an organism. They require a minimum number of clones to ensure all genes are captured. cDNA libraries are made from mRNA and represent expressed genes, avoiding introns. Key steps in making cDNA libraries include mRNA isolation, cDNA synthesis, addition of linkers, and ligation into a vector. Screening methods to identify clones of interest include hybridization, expression screening, and hybrid arrest/release.
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
The three hybrid system of yeast has been described in this ppt. Yeast one Hybrid system, yeast two hybrid system and yeast 3 hybrid system is explained. This explain about the DNA-protein interaction and Protein-DNA-Protein interaction.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.
The three hybrid system of yeast has been described in this ppt. Yeast one Hybrid system, yeast two hybrid system and yeast 3 hybrid system is explained. This explain about the DNA-protein interaction and Protein-DNA-Protein interaction.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
STS stands for sequence tagged site which is short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
This pdf is about the DNA Libraries / Genomic DNA vs cDNA.
For more details visit on YouTube; @SELF-EXPLANATORY; https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos Thanks...!
DNA SEQUENCING METHODS AND STRATEGIES FOR GENOME SEQUENCINGPuneet Kulyana
This presentation will give you a brief idea about the various DNA sequencing methods and various strategies used for genome sequencing and much more vital information related to gene expression and analysis
CLONING METHODOLOGIES:(GUYS LEARN CLONING IN EASIER WAY)
■PRINICIPLES AND STEPS INVOLVED IN CLONING
■METHODS INVOLVED IN cDNA OR GENOMIC CLONING
1.Isolation of mRNA
2.Synthesis of first strand of cDNA
3.Synthesis of second strand of cDNA
4.Cloning of cDNA
5.Introduction into Host Cell
6.Clone Selection
■OTHER TECHNIQUES INVOLVED IN CLONING OR FOREIGN GENE TRANSFER.
■EXPRESSION CLONING AND PROTEIN-PROTEIN INTERACTIONS
■cDNA or GENOMIC DNA LIBRARY CONSTRUCTION
■SIMILARITIES BETWEEN cDNA AND GENOMIC DNA LIBRARY
■ADVANTAGES AND DISADVANTAGES OF cDNA AND GENOMIC LIBRARIES
■REFERENCES.
Make my ppt useful in research and it also helpful for student's for Notes.
DNA Libraries are collection of fragments of DNA cloned to a vector so that researchers can easily identify and isolate a particular gene of interest for future use.
This is a presentation by Dada Robert in a Your Skill Boost masterclass organised by the Excellence Foundation for South Sudan (EFSS) on Saturday, the 25th and Sunday, the 26th of May 2024.
He discussed the concept of quality improvement, emphasizing its applicability to various aspects of life, including personal, project, and program improvements. He defined quality as doing the right thing at the right time in the right way to achieve the best possible results and discussed the concept of the "gap" between what we know and what we do, and how this gap represents the areas we need to improve. He explained the scientific approach to quality improvement, which involves systematic performance analysis, testing and learning, and implementing change ideas. He also highlighted the importance of client focus and a team approach to quality improvement.
Ethnobotany and Ethnopharmacology:
Ethnobotany in herbal drug evaluation,
Impact of Ethnobotany in traditional medicine,
New development in herbals,
Bio-prospecting tools for drug discovery,
Role of Ethnopharmacology in drug evaluation,
Reverse Pharmacology.
The Indian economy is classified into different sectors to simplify the analysis and understanding of economic activities. For Class 10, it's essential to grasp the sectors of the Indian economy, understand their characteristics, and recognize their importance. This guide will provide detailed notes on the Sectors of the Indian Economy Class 10, using specific long-tail keywords to enhance comprehension.
For more information, visit-www.vavaclasses.com
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
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It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
How libraries can support authors with open access requirements for UKRI fund...
construction of genomicc dna libraries
1.
2. ContentsContents
Genomic librariesGenomic libraries
Representative gene libraries, Size of library,
Genomic DNA, Vectors
cDNA librariescDNA libraries
mRNA isolation, purification and fractionation,
Synthesis of cDNA, Treatment of cDNA, Ligation
to vector
Screening proceduresScreening procedures
Screening, Colony and plaque hybridization,
Expression screening, Hybrid arrest and release,
Chromosome walking
3. Genomic libraries —Genomic libraries —
Representative gene librariesRepresentative gene libraries
Genomic libraryGenomic library:: A collection of different DNA
sequence from an organism each of which has been
cloned into a vector for ease of purification, storage
and analysis .
Genomic librariesGenomic libraries
cDNA librariescDNA libraries
Gene library
(made from genomic DNA)
(made from cDNA- copy of mRNA)
4. Important consideration:
Making a representative library---
Containing all the original sequences
(1) Lacking restriction sites
(2) Does not contain sufficient clones
(3) Enrich certain sequences, lack others
Missing original sequence:
Too long for the vector used
5. Genomic libraries —Genomic libraries — Size of librarySize of library
The formula to calculate the number of recombinants:
P: desired probability
f : the fraction of the genome in one insert
6. For a probability of 0.99 with insert sizes of 20kb
these values for the E. coli (4.6×106 bp) and human
(3×109 bp) genomes are :
Easy to make good genomic libraries from
prokaryotes in plasmids where the insert size is 5-
10kb, as only a few thousand recombinants will be
needed.
7. Genomic libraries —Genomic libraries — Genomic DNAGenomic DNA
Purify genomic DNA
Correct size for cloning into the chosen vector:
Physical shearing and restriction enzyme digestion
EukaryotesEukaryotes
ProkaryotesProkaryotes
Clone the fragments into vectors
8. 1. Purification of genomic DNA
Remove protein, lipids and other unwanted
macro-molecules by protease digestion and
phase extraction ( Phenol-chloroform ) .
EukaryotesEukaryotes:: Prepare cell nuclei (fractionation, reduce
contamination from organelle DNA)
Prokaryotes:Prokaryotes: Extracted DNA directly from cells
9. 2. Break DNA into fragments randomly
(1) Physical shearing
Pipeting, mixing or sonication. The
choice of method and time of exposure
depend on the size requirement of the
chosen vector.
10. (2)Restriction enzyme digestion
Partial digestion:
Get a greater lengths of DNA fragments. Time of
digestion and ration of restriction enzyme to DNA
are dependent on the desired insert size range, the
DNA is not digested at every recognition sequence
that is present.
Sau3A: 5’-/GATC-3’,
BamH1: 5’-G/GATCC-3’
11. Ends produced (sticky or blunt) &
the cleaved ends of the vector to be
cloned
DNA modifications
Whether the enzyme is inhibited by DNA
modifications (CpG methylation in
mammals).
12. I1 Genomic libraries —I1 Genomic libraries — VectorsVectors
According to genome’s size, select a proper
vector to construct a library .
Vectors Plasmid phageλ cosmid YAC
insert (kb) 10 23 45 1000
13. λ replacement vector
2.Ligation
3. Packing with a mixture of
the phage coat proteins and
phage DNA-processing
enzymes
4. Infection and
formation of plaques
1.Preparation of arms and
genomic inserts
Library constructed
14. cDNA libraries —cDNA libraries — mRNA isolation,mRNA isolation,
purification and fractionationpurification and fractionation
The most commonly chosen genomic cloning vectors are λ
replacement vectors which must be digested with restriction
enzymes to produce the two λ end fragment or λ arms between
which the genomic DNA will be ligated.
1. Characteristics of cDNA libraries
2. Methods to isolate mRNA
3. Check the mRNA integrity
4. Cloning the particular mRNAs
15. 1. Characteristics of cDNA libraries
(a) No cDNA library was made from prokaryotic
mRNA.
• Prokaryotic mRNA is very unstable
• Genomic libraries of prokaryotes are easier to make and
contain all the genome sequences.
(b) cDNA libraries are very useful for eukaryotic
gene analysis
• cDNAs represent the transcribed parts of the genome
(i.e. the genes rather than the nontranscribed DNA).
cDNAs have no introns → genes can be expressed in E.
coli directly
• Tissue or cell type specific (differential expression of
genes
16. mRNA isolation, purification
Check the RNA integrity
Fractionate and enrich mRNA
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector
2. Methods to isolate mRNA
17. • Most eukaryotic mRNAs are polyadenylated
at their 3’ ends.
• oligo (dT) can be bound to the poly(A) tail and
used to recover the mRNA.
3’-AAAAAAAAAAn5’- cap
18. (1)Traditional method was done by pass a preparation
of total RNA down a column of oligo (dT)-cellulose.
(2)More rapid procedure is to add oligo(dT) linked to
magnetic beads directly to a cell lysate and ‘pulling
out’ the mRNA using a strong magnet.
(3)Lying cells and then preparing mRNA-ribosome
complexes on sucrose gradients.
Three methods:
20. Make sure that the mRNA is not degraded.
Methods:
(1)Translating the mRNA : use cell-free translation
system as wheat germ extract or rabbit reticulocyte
lysate to see if the mRNAs can be translated
(2)Analysis the mRNAs by gel electrophoresis: Use
agarose or polyacrylamide gels
3. Check the mRNA integrity
21. 4. Cloning the particular mRNAs
Is useful especially one is trying to clone a
particular gene rather to make a complete cDNA
library
• Fractionate on the gel: Performed on the basis of size, mRNAs
of the interested sizes are recovered from agarose gels
•Enrichment: Carried out by hybridization.
•Example: make a cDNA library of the mRNA sequences that
are induced with a hormone (hybridization , substrated cDNA
library)
22. I2 cDNA libraries —I2 cDNA libraries — Synthesis of cDNASynthesis of cDNA
• The first strand and Second strand synthesis
23. cDNA libraries —cDNA libraries — Treatment of cDNATreatment of cDNA
Blunt end ligation of large fragments is not efficient,
so we have to use special linkers to create sticky ends
for cloning.
DNA linker:
DNA adaptor:
HO-CCGAATTCGGGGGG
3’-GGCTTAAGCCCCCC
HO - CGGGGGG
3’-TTAAGCCCCCC
24.
25. The process :
Move protruding 3’-ends( strand-special
nuclease)
Fill in missing 3’ nucleotide( Klenow fragment of
DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers( T4 DNA ligase)
Restriction enzyme digestion( EcoRI )
26. cDNA libraries —cDNA libraries — Ligation to vectorLigation to vector
Any vectors with an EcoRI site would suitable
for cloning the cDNA.
The process :
Dephosphorylate the vector
Ligate vector and cDNA with T4 DNA ligase
Plasmid or λ phage vector, short, plasmid vector;
cDNA libraries, λ phage vector;
λgt11 has EcoRI site placed near the C terminus of its
lacZ gene, enabling expression of the cDNA as part of a
large β-galactosidase fusion protein.
27. Screening procedures —Screening procedures — ScreeningScreening
Screening: The process of identifying one particular
clone containing the gene of interest from among
the
very large number of others in the gene library .
(1) Using nucleic acid probe to screen the library
based on hybridization with nucleic acids.
(2) Analyze the protein product
28. Screening libraries
Hybridization to identify the interested DNA or its RNA product.
(1) DNA radiolabeled probes which is
complementary to a region of the interested
gene.
29. DNA sequence information:
An oligonucleotide derived from the sequence of a
protein product of the gene.
A DNA fragment/oligo from a related gene of another
species.
Preparation methods:
Automated chemical synthesis (short probes)
PCR
30. Screening procedures —Screening procedures —
Colony and plaque hybridizationColony and plaque hybridization
Transfer the DNA in the plaque or colony to a Nylon or
nitrocellulose membrane
Phage DNA bind to
the membrane directly
Bacterial colonies must be lysed to
release DNA on the membrane surface.
(Alkali treatment)
Hybridization (in a solution
containing Nucleic acid probe)
Wash to remove unhybri-
dized probe and visualize
X-ray film (radio-
actively labeled )
Antibody or enzyme
(modified nucleotide
labeled)
Line up the hybridizated region or
repeated hybridization
31. Transfer to nitrocellulose
or nylon membrane
Denature DNA
(NaOH). Bake
onto membrane
Probe with 32
p-labled DNA
complementary to
gene of interest
Expose to film
Select positive
from master plate
Keep master
Plate
Screening by plaque hybridization
32.
33. Screening procedures —Screening procedures —
Expression screeningExpression screening
If the inserts are cloned into an expression
sites, it may be expressed. Therefore, we can
screen for the expressed proteins.
Example: the EcoRI site of lgt11 vector. The
inserted genes have one in six possibilities
(1/6) to be in both the correct orientation (two
possibilities; ) and reading frame (three
possibilities).
34. Screening procedures —Screening procedures —
Hybrid arrest and releaseHybrid arrest and release
(1) Hybrid arrested translation
Individual cDNA clones or pools of clones can be used to
hybridize to mRNA preparation.
Translate the mRNA population directly, and the
inhibition of translation of some products detected.
( 2 ) Hybrid release translation
Purify the hybrids and the hybridized mRNAs
released from them and translated, it identifies the
protein encoded by the cDNA clone
35. Screening procedures —Screening procedures —
Chromosome walkingChromosome walking
Definition: To clone the desired gene by
repeated isolating adjacent genomic clones
from the library.
36.
37. Multiple choice questionsMultiple choice questions
1. Which two of the following statements about genomic libraries are false?
A genomic libraries are made from cDNA.
B genomic libraries must be representative if they are to contain all the genes in an
organism.
C genomic libraries must contain a minimum number of recombinants if they are to
contain all the genes In an orgamsm.
D the DNA must be fragmented to an appropriate size for the vector that is used.
E genomic libraries made from eukaryotic DNA usually use plasmid vectors.
2. Which statement correctly describes sequential steps in cDNA cloning?
A reverse transcription of Mrna second strand synthesis cDNA end modification ligation to
vector.
B mRNA preparation cDNA synthesis using reverse transcriptase second strand synthesis
using terminal transferase, ligation to vector.
C mRNA synthesis using RNA polymerase reverse transcription of mRNA, second strand
synthesis, ligation to vector.
D double stranded cDNA synthesis restriction enzyme digestion addition of linkers
ligation to vector.
38. 3. Which one of the following is not a valid
method of screening a library?
A hybridization of colony / plaquelifted DNA
using a nucleic acid probe.
B using antibodies raised against the protein of
interest to screen an expression library.
C screening pools of clones from an expression
library for biological activity.
D hybridization of colony/plaquelifted DNA
using an antibody probe.