cDNA Library. MB206. Making a DNA library . Types of Libraries. (3’UTR). (5’UTR). Genomic DNA libraries. -Contains the whole genome of an organism. -A restriction-enzyme is used to cut the genome (the DNA) at numerous locations. . polyA. cDNA. Reverse transcribe (and more). polyA.
-Contains the whole genome of an organism.
-A restriction-enzyme is used to cut the genome (the DNA) at numerous locations.
[mRNA it is used to create it]cDNA Libraries
Figure 7-14, 7-15
Consideration: Try not to cut the gene or operon of interest
A ‘four-base cutter’ recognition sequence would occur once every 44 = 256 bp
A ‘six-base cutter’ would give you fragments of about 4000 bp
An ‘eight-base cutter’ recognition sequence would occur once every 48 or 65,536 bp.
Up to 100-150kbp inserts
Up to 500kbp inserts
Lodish, et al. Fig 7-12
Lodish, et al. Fig 7-1
Make sure that the mRNA is not degraded.
Translating the mRNA :
use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translated
Analysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels
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 gels
Enrichment:carried out by hybridization
Example: clone the hormone induced mRNAs (substrated cDNA library)
First stand synthesis:
materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1.1)
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.
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
Klenow polymerase or reverse
Transcriotase Four dNTPs
Single strand-specific nuclease
treat with E.coRI methylase
Add E.colRI linkers
using T4 DNA ligase
Ligate to vector and transfom
Fig2.1 Second strand synthesis
Blunt 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)
Tailing with terminal transferase or using adaptor molecules
Restriction enzyme digestion(E.coRI )
Any vectors with an EcoRI site would suitable
for cloning the cDNA.
The process :
Dephosphorylate the vector with alkaline
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .
Searching the genes of interest in a DNA library
Transfer the DNA in the plaque or colony to a
Nylon 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)
actively labeled )
antibody or enzyme
(modified nucleotide labeled
Wash to remove unhybri-
dization probe and visualize
Line up the hybridizated region or
or nylon membrane
from master plate
Bake onto membrane
Probe with 32p-labled DNA
gene of interest
Expose to film
Screening by plaque hybridization