DNA Libraries are collection of fragments of DNA cloned to a vector so that researchers can easily identify and isolate a particular gene of interest for future use.
1. S R U T H Y K . S
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VPB 321
DNA LIBRARIES
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.
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.
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.
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.
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
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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)
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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.
