SRUTHY K . S11-BVP-216

VPB 321

DNA LIBRARIES

DNA LIBRARY

The term "library" can refer to a population of organisms, each of which carries a DNA molecule inserted into a cloning vector, or alternatively to the collection of all of the cloned vector molecules.

Collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study.

Genomic libraries

cDNA libraries

Gene library

(made from genomic DNA)

(made from cDNA- copy of mRNA)

For the construction of DNA library

Size of the gene

Capacity of the vector

Molecular tools

Vectors

Vector type Max. Insert size cloned DNA

(kb)

Approx.no: of cloned DNA required in a

library

Plasmid 20 >10^5

Lambda phage 20 5 x10^5

Cosmid 45 2 x 10^5

BAC >500 5 x 10^4

YAC 1Mb 1o^5

Genomic DNA library: Contains the whole genome of an organism

Genome size is expressed in terms of number of base pairs.

Smallest genomic size is…………..????

For human the genomic size is………….????

STEPS

1) Purification of genomic DNA

PROKARYOTES

EUKARYOTES

•Genomic libraries of prokaryotes are easier to make and contain all the genome sequences.

2) Cleaving the genome into smaller fragments by restriction endonucleases.

• Parial digestion is used to getting longer DNA sequences.

3) In cooperation of these fragments into a suitable vector.

4) Introduction of vector into a suitable host like bacterium(Transformation)

Multiplication

Screening

Screening: The process of identifying one particular clone containing the gene of interest from amongthe very large number of others in the gene library .

Expression screening

Hybridisation technique

PCR

Expression screening

Eg: Finding the gene for alanine production

Grow in alanine deficit medium

Then they are labelled in the petri plate indicates that gene for alanine production is stored in bacteria.

Hybridisation technique

cDNA Library

No cDNA library was made from prokaryotic mRNA…..???

cDNA libraries are very useful for eukaryotic gene analysis.

mRNA isolation, purification

Check the RNA integrity

Synthesis of cDNA

Treatment of cDNA ends

Ligation to vector

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

Check the mRNA integrity

Make sure that the mRNA is not degraded.

•Translating the mRNA

•Analysis the mRNAs by gel elctrophoresis

Synthesis of cDNA

First stand synthesis: 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.

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’

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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

Fig2.1 Second strand synthesis23

Treatment of cDNA ends

Blunt and ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.

Move protruding 3’-ends (strand-special nuclease)

Fill in missing 3’ nucleotide

Ligate the blunt-end and linkers(T4 DNA ligase)

Restriction enzyme digestion (E.coRI )

Tailing with terminal transferase or using adaptor molecules

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Ligation to vector

Any vectors with an EcoRI site would suitablefor cloning the cDNA.

Dephosphorylate the vector with alkalinephosphatase

Ligate vector and cDNA with T4 DNA ligase

(plasmid or λ phage vector)

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Uses of cDNA library

Used when reproducing eukaryotic genomes as the amount of information is reduced to remove the large number of non-coding regions from the library.

To express eukaryotic genes in prokaryotes.

Useful for subsequently isolating the gene that codes for that mRNA.

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