This document describes DNA libraries and the process of creating them. It discusses genomic libraries, which contain an organism's entire genome, and cDNA libraries, which contain copies of mRNA. The key steps to create a library are: 1) isolating and fragmenting DNA, 2) cloning the fragments into vectors, 3) transforming the vectors into host cells, 4) multiplying and screening the clones to identify fragments of interest. cDNA libraries are useful for studying eukaryotic genes as they remove non-coding regions.

1. DNA LIBRARY
By
Anita Devi P.hD
15099009

2. 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.

3. Genomic libraries
cDNA libraries
Gene
library
(made from genomic DNA)
(made from cDNA- copy of mRNA)

4. For the construction of DNA library
 Size of the gene
 Capacity of the vector
 Molecular tools
 Vectors

5. 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

6.  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………….????

7. STEPS
1) Purification of genomic DNA
PROKARYOTES
EUKARYOTES
•Genomic libraries of prokaryotes are
easier to make and contain all the genome
sequences.

8. 2) Cleaving the genome into smaller fragments
by restriction endonucleases.
• Parial digestion is used to getting longer DNA
sequences.

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

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

11.  Multiplication
 Screening

12.  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 .
 Expression screening
 Hybridisation technique
 PCR

13. 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.

14. Hybridisation technique

16. cDNA Library
 No cDNA library was made from prokaryotic
mRNA…..???
 cDNA libraries are very useful for eukaryotic gene
analysis.

18. mRNA isolation, purification
Check the RNA integrity
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector

19. 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

20. Check the mRNA integrity
 Make sure that the mRNA is not degraded.
•Translating the mRNA
•Analysis the mRNAs by gel
elctrophoresis

21. 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.

22. 5’ mRNA AAAAA-3’
HO-TTTTTP-5’
5’
Reverse transcriptase
Four dNTPs
AAAAA-3’
TTTTTP-5’
mRNA
mRNA
cDNA
cDNA
cDNA
Duplex cDNA
AAAAA-3’
TTTTTP-5’
TTTTTP-5’
3’
3’-CCCCCCC
Terminal transferase
dCTP
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
Klenow polymerase or reverse
Transcriotase Four dNTPs
5’-pGGGG-OH
5’
3’-CCCCCCC
5’-pGGGG
3’-CCCCCCC TTTTTP-5’
-3’
Angelia 09 22

23. 5’-pGGGG
3’-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

24. 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
24

25. Ligation to vector
Any vectors with an EcoRI site would suitable
for cloning the cDNA.
Dephosphorylate the vector with alkaline
phosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
25

26. 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.