dna library lecture-gene libraries and screening

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


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)


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


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)


Identify the protein product of an interested gene

1.Protein activity2.Western blotting using a

specific antibody




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



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.


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


I3-5 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 .


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.


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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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dna library lecture-gene libraries and screening

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