DNA library is a collection of DNA, three types of recombinant DNA libraries are present 1) Genomic library 2) Chromosomal library 3) Complementary DNA library

1. DNA Libraries - Definitions
DNA library (a collection of DNA clones):
It is a convenient storage mechanism of genetic information.
Recombinant DNA Libraries (3 types):
1. Genomic library, Collection of cloned restriction enzyme digested
DNAs containing at least one copy of every DNA sequence in a
genome.
2. Chromosome library, Collection of cloned restriction enzyme
digested fragments from individual chromosomes.
3. Complementary DNA (cDNA) library, Collection of clones of DNA
copies made from mRNA isolated from cells.

2. Genomic libraries
• Goal is to fragment a particular genome (e.g. human) into useful
sized pieces and to have a mechanism whereby each piece can be
isolated, identified and manipulated.
• One essential manipulation is ability to replicate fragment for further
use and study.
• Fragmentation of a genome can be accomplished using an
appropriate restriction endonuclease.
Commonly used vectors
– Bacteriophage lambda
– Cosmids
– YACs may also be used as vectors
• Human Genome Project (HGP)
– Entire human genome has been sequenced (April 2000)
– Project began in 1990 – Joint Venture
• Human Genome Organization (HuGO) (USA, UK, France,
Japan mainly)
• CELERA

3. Fig. 7.9 Partial digestion with Sau3A
Results in a library of overlapping DNA
fragments of various sizes.

4. DNA library

5. Table 4.4. Sizes of human genomic libraries prepared in different types of cloning
vector
Number of clones*
Type of vector Insert size (kb) P = 95% P = 99%
λ replacement 18 532 500 820 000
Cosmid, fosmid 40 240 000 370 000
P1 125 77 000 118 000
BAC, PAC 300 32 000 50 000
YAC 600 16 000 24 500
Mega-YAC 1400 6850 10 500
Calculated from the equation
Table 4.4. Sizes of human genomic libraries prepared in
different types of cloning vector
• N = ln (1-P)/ ln (1-f)
– N = Number of required clones
– P = To screen for a clone in a library usually want a 99%
probability that your clone is found there (P= 0.99)
– f = Frequency is the size of the DNA fragment in the library/the
size of the haploid genome

6. Finding size of a library
 Number of clones required for a complete library can be calculated
from;
- the size of the genome and
- average size of overlapping fragments cut by restriction enzymes
 Library should contain many times more clones than the calculated
minimum number of clones.

8. cDNA libraries
• cDNA libraries are used to detect or sequence genes for proteins
(cDNAs are generated for genes that are transcribed!)
Bacteriophage λ insertion vectors or plasmids are used for cloning
• Choice depends upon:
– Abundance of mRNA
– Size of desired library
– Screening method
Disadvantages of cDNA Library
1. cDNA is derived from mature mRNA, does not include introns
2. cDNA may contain less information than the coding region
3. cDNA library reflects gene activity of a cell at the time mRNAs are
isolated (varies from tissue to tissue and with time)
4. mRNA degrades quickly

9. Creating a cDNA library 3-steps:
1. Isolate mRNA
2. Synthesize cDNA
3. Clone cDNA
• Anneal a short oligo dT (TTTTTT) primer to the poly-A tail.
• Primer extended by reverse transcriptase 5’ to 3’ creating a
mRNA-DNA hybrid.
• mRNA is next degraded by Rnase H, but leaving small RNA
fragments intact to be used as primers.
• DNA polymerase I synthesizes new DNA 5’ to 3’ and removes
the RNA primers.
• DNA ligase connects the DNA fragments.
• Result is a double-stranded cDNA copy of the mRNA.

12. Chromosomal Library
– Series of clones that contain
overlapping pieces of
chromosomal DNA
Overlapping regions allow researchers
to identify their order along the
chromosome

13. Strategies for Library Screening
Of thousands of clones in a genomic or cDNA library, which one is
one you are interested in?
There must be some DNA sequence information to identify it
Selection of recombinant clones necessitates the use of an
appropriate selectable marker system
-Screening by vector molecules: using antibiotic resistance genes
or gene complementation
- Generalized recombinant screening: by insertional inactivation
-Directed recombinant screening: This can be achieved
Hybridization-based screening
Hybridization probes
Radioactive labeled probes
Biotinylated probes
PCR-based screening

14. Screening a genomic library

15. 15
Identifying a cloned gene
by Colony hybridization
– Master plate with bacterial
cells containing
recombinant vector with
different pieces of
chromosomal DNA
– Use single-stranded DNA
probe
– Dark spots correspond to
colonies with gene of
interest

17. Screening a cDNA library
with antibody probe

18. lambda expression cloning to identify a
cloned DNA based on binding of the
encoded protein to a specific antibody

19. Phage Display
Involves cloning cDNA into phage vectors & expressing foreign
proteins on phage surface as fusion Protein. Purification of
recombinant is done via biotin-streptavidin based system