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Genomic libraries
cDNA libraries
Screening procedures
Gene libraries and screening
Introduction
• The use of genetic information is a powerful tool that today is becoming more readily available to scientists.
• In order to use this powerful tool it necessary to know how to navigate throughout the entire genome. The human genome is about 3 x 10E9 bp.
• In humans this project is known as Human Genome Project.
Gene 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 libraries
cDNA libraries
Gene library (made from genomic DNA)
(made from cDNA- copy of mRNA)
I1 Genomic
libraries
Size of library (ensure enough clones)
must contain a certain number of recombinants for there to be a high probability of it containing any particular sequence The formula to calculate the number of recombinants:
N = ln (1-P)
ln (1-f)
P: desired probability f : the fraction of the genome in one insert
I1 Genomic
libraries
For example :for a probability of 0.99 with insert sizes of 20 kb these values for the E.coli (4.6×106 bp) and human (3×109 bp) genomes are :
N E.coli= = 1.1 ×103 ln( 1-0.99)
ln[1-(2×104/4.6×106)]
Nhuman= = 6.9 ×105 ln(1-0.99)
ln[1-(2 ×104/3 ×109)]These values explain why it is possible 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.
I1 Genomic
libraries
Genomic DNA libraries
Purify genomic DNA
Fragment this DNA : physical
shearing and restriction enzyme
digestion
eukaryotes
prokaryotes
Clone the fragments into vectors
I1 Genomic
libraries
To make a representative genomic libraries ,genomic DNA must be purified and then broken randomly into fragments that are correct in size for cloning into the chosen vector.
Purification of genomic DNA :
Prokaryotes :extracted DNA directly from cells
remove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction.
Eukaryotes :prepare cell nuclei
I1 Genomic
libraries
Break DNA into fragments randomly:
Physical shearing : pipeting, mixing
Restriction enzyme digestion: partial digestion is
preferred to get a greater lengths of DNA fragments.
I1 Genomic
libraries
Sau3A: 5’-/GATC-3’, less selectivityBamH1: 5’-G/GATCC
Selection of restriction enzyme1. Ends produced (sticky or blunt) &
The cleaved ends of the vector to be cloned
2. Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals
3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.
I1 Genomic
libraries
Generating A Genomic Library
• λ-phage is treated with restriction enzymes that produce λ arms with sticky end. These arms contain all the lytic genetic information that is needed for replication and produces room for insertion of new genetic information.
• DNA sequence is obtain from the cell of interest. It is cleaved with restriction enzymes that produce 20kb fragments that have complementary sticky ends.
• Both are mixed in equal amounts and are treated with a DNA ligase that cleaves them together.
• Afterward the entire combined sequence is packed to the phage head.
λ-phage as a Vector
• The genomic library is generated by using λ-phage for the following reasons.
1. A large number of λ phage can be screened simultaneously (5 x 10E4 phage plagues).
2. λ phage as a higher transformation efficiency about 1000 times higher compared to a plasmid.
• The vector as to maintain its lytic growth.
• Lysogenic pathway and other viral genes that are not important are replaced with the DNA to be cloned.
λ-phage as a Vector (Cont.)
• An infected E.Coli will produce what are know as concatomers (which is the viral genome) on either site of the concatomers there is a site called COS Site.
• Two proteins recognize this site A protein and Nu protein, which will lead to the insertion of the λ DNA into the phage head. The chromosomal DNA that lacks the COS sites will not enter the phage head. Once the genetic information is inserted the tail will assemble.
• A 50kb can be inserted into the phage.
Packaging of the Recombinant DNA• To prepare the phage an E.coli cell is infected with a mutant λ-phage
that as a defective “A-protein” (which is one of two genes that are responsible for packaging genetic information).
• Therefore the E.Coli accumulates empty heads and also preassembled tails.
• Once enough heads and tails are assembled we lysate the E.Coli cells.• To the mixture of heads and tail we add isolated A protein (obtained
from E.Coli infected with λ-phage). • In the next step we add the recombinant DNA that has the λ-phage
genetic information (which also includes COS sites). • At this point we have a mixture containing mutant λ-phage heads and
tails. There is isolated A protein and recombinant DNA containing λ-phage genetic information with COS sites.
• Therefore we have all the components necessary to package the recombinant DNA into the λ-phage head. Once the information is inserted the tail assembles and we have an infectious phage that contains the recombinant DNA sequence.
Vectors
According to genome’s size,we can select a proper vector to construct a library .
Vectors Plasmid phageλ cosmid YAC
insert (kb) 5 23 45 1000
The most commonly chosen genomic cloning vectors are λ relacement 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 digested
I1 Genomic
libraries
cos cos
Long (left)arm
short (right)arm
Exogenous DNA(~20-23 kb)
λ phage vector in cloning
cos cos
Long (left)arm
short (right)arm
Exogenous DNA(~20-23 kb)
λ replacement vector cloning
2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes 3. Infection and
formation of plaques
Library constructed
1. Ligation
0.preparation of arm and genomic inserts
I cDNA libraries mRNA isolation, purification
Check theRNA integrity
Fractionate and enrich mRNA
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector
Gene libraries and screening
cDNA libraries
1.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.
I 2 cDNA libraries
2.cDNA libraries are very useful for eukaryotic gene analysis
• Condensed protein encoded gene libraries, have much less junk sequences.
• cDNAs have no introns genes can be expressed in E. coli directly
• Are very useful to identify new genes• Tissue or cell type specific (differential
expression of genes)
cDNA libraries
I 2 cDNA libraries
mRNA isolation
• 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.
AAAAAAAAAAn5’ cap
I 2 cDNA libraries
I 2 cDNA libraries
1.Traditionally 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.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradients
Three methods to isolate mRNA.
I2 cDNA libraries
Make sure that the mRNA is not degraded. Methods:Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translatedAnalysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels
Check the mRNA integrity
I2 cDNA libraries
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 gelsEnrichment: carried out by hybridizationExample: clone the hormone induced mRNAs (substrated cDNA library)
I2 cDNA libraries
Synthesis of cDNA :
First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs Second strand synthesis: best way of making full-length cDNA is to ‘tail’ the 3’-end of the first strand and then use a complementary primer
to make the second.
I2 cDNA libraries
5’ mRNA AAAAA-3’ HO-TTTTTP-5’
5’
Reverse transcriptaseFour dNTPs
AAAAA-3’TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
AAAAA-3’
TTTTTP-5’
TTTTTP-5’
3’
3’-CCCCCCC
Terminal transferasedCTP
Alkali (hydrolyaes RNA)Purify DNA oligo(dG)
Klenow polymerase or reverseTranscriotase Four dNTPs
5’-pGGGG-OH
5’
3’-CCCCCCC
5’-pGGGG3’-CCCCCCC TTTTTP-5’
-3’
The first strand synthesis
I2 cDNA libraries
5’-pGGGG3’-CCCCCCC
HO-CCGAATTCGGGGGG 3’-GGCTTAAGCCCCCC
5’-pAATTCGGGGGG
TTTTTGGCTTAAGCC-OH CCGAATTCGG-3’
3’-CCCC
3’-CCCCCCC
3’-CCC 5’-pGGGG
5’-pGGGG
TTTTTp-5’ -3’
TTTTTp-5’
TTTTTp-5’
-3’
-3’
TTTTTGGCTTAAp-5’
HO-CCG/AATTCGG-3’ 3’-GGCTTAA/GCC-OH
CCG-3’
Duplex cDNA
Single strand-specific nuclease
Klenow polymerase
treat with E.coRI methylase
Add E.colRI linkers using T4 DNA ligase
E.colRI digestion
Ligate to vector and transfom
Second strand synthesis
Treatment of cDNA endsBlunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.
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 (E.coRI )
Tailing with terminal transferase or using adaptor molecules
I2 cDNA libraries
Ligation to vector Any vectors with an E.coRI site would suitablefor cloning the cDNA.
The process :
Dephosphorylate the vector with alkalinephosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
I2 cDNA libraries
Screening procedures Screening
Colony and plaque hybridization
Expression screening
Hybrid arrest and release
Chromosome walking (repeat screening)
Gene libraries and screening
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.
I3 Screening procedures
Screening libraries
Hybridization to identify the interested DNA or its RNA product
1. Radiolabeled probes which is complementary to a region of the interested geneProbes: • An oligonucleotide derived from the
sequence of a protein product of the gene• A DNA fragment/oligo from a related gene
of another species 2. Blotting the DNA or RNA on a membrane 3. Hybridize the labeled probe with DNA
membrane (Southern) or RNA (Northern) membrane
Searching the genes of interest in a DNA library
I3 Screening procedures
Colony and plaque hybridization
Transfer the DNA in the plaque or colony to aNylon or nitrocellulose membrane
Phage DNA bind to the membrane directly
Bacterial colonies must be lysed to release DNA on the membrane surface.
Hybridization (in a solution Containing Nucleic acid probe)
Wash to remove unhybri-dization probe and visualize
X-ray film(radio-actively labeled )
antibody or enzyme(modified nucleotide labeled
Line up the hybridizated region orrepeated hybridization
(Alkali treatment)
I3 Screening procedures
Identify the protein product of an interested gene
1.Protein activity2.Western blotting using a
specific antibody
I3 Screening procedures
Expression screening
Expression screening
If the inserts are cloned into an expression sites, it may be expressed. Therefore, we can screen for the expressed proteins. However, this screening may miss the right clone
I3 Screening procedures
Expression screening
The procedure
‘Plaque lift’ ( taken by placing a membrane on the dish of plaque)
Immersed in a solution of the antibody
Detected by other antibodies
Repeat cycles of screening to isolate pure plaques
Antibodies can be used to screen the expression library.
I3 Screening procedures
Hybrid arrest and screen
Individual cDNA clones or pools of clones can be used to hybridize to mRNA preparation
Hybrid arrest :translate the mRNA population directly, and the inhibition of translation of some products detected.
Hybrid release translation : purify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone
I3 Screening procedures
I3-5 Chromosome walking
Definition: To clone the desired gene by repeated isolating adjacent genomic clones from the library.
to obtain overlapping genomic clones that represent progressively longer parts of a particular chromosome .
I3 Screening procedures
Process:
1. Prepare a probe from the end insert .
2.The probe are used to re-screen the library by colony or plaque hybridization
3.Analyzed the new isolate clones and posited them relative to the starting clone. some will be overlapping.
4. Repeated the whole process using a probe from the distal end of the second clone.
I3 Screening procedures
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Vector arm Genomic clone insert Vector arm
Prepare probe from ends of insert
Re-screen genomic library
Restriction
Restriction map new genomic clones
Prepare new probes from distal ends of least overlapping insert.Re-screen genomic library . Restriction map new genomic clones
Chromosome walking
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