Transcript cDNA Library

cDNA Library
MB206
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Making a DNA library
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Types of Libraries
Genomic Library
• whole genes w/ promoters & introns (Euk.),
operons (bacteria), DNA regulatory elements…
cDNA Library
• mRNA transcript only w/ 5’ & 3’ untranslated
regions (UTRs), no introns, tissue specific.
(5’UTR)
(3’UTR)
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Genomic DNA libraries
-Contains the whole genome of an
organism.
-A restriction-enzyme is used to
cut the genome (the DNA) at
numerous locations.
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Genomic Libraries
cDNA library
Genomic DNA
mRNA
polyA
polyA
Reverse
transcribe
cDNA
(and more)
polyA
Genomic DNA library
Clone in
vector
Genomic DNA
Digest
DNA fragments
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cDNA Libraries
cDNA library
Genomic DNA
mRNA
polyA
Reverse
transcribe
cDNA
(and more)
polyA
polyA
Genomic DNA library
Clone in
vector
Genomic DNA
Digest
DNA fragments
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cDNA Libraries

“complementary” DNA
[mRNA it is used to create it]
Purify mRNA

mRNA-> single stranded cDNA using reverse
transcriptase.

Single stranded cDNA -> double stranded cDNA (DNA
polymerase and other “cloning tricks”).

Linkers added to cDNA & clone into vectors as seen in
genomic DNA libraries
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Lodish, et al. 1999
Figure 7-14, 7-15
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Genomic Libraries
“The Details”
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Genomic Libraries
cDNA library
Genomic DNA
mRNA
polyA
Reverse
transcribe
cDNA
(and more)
polyA
polyA
Genomic DNA library
Clone in
vector
Genomic DNA
Digest
DNA fragments
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Digest genomic DNA with restriction enzymes

Which restriction enzyme should
we select?
Consideration: Try not to cut the gene
or operon of interest

V. fischeri LUX operon 9kb
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Selecting a restriction enzyme

What is the average size fragment
that any given enzyme will yield?

If enzyme recognizes 6bp, statistically
any given 6bp sequence will appear
randomly every 46 bases (every 4096
bases)
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How often does the enzymes cut?
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.
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How often do enzymes really
cut?

But also we need to consider G+C content
of the genome:
 V.
fischeri G+C content = 40%
 Sal
I recognizes sites that are G+C
rich (GTCGAC)  it will cut less often
in Vibrio genomic DNA.
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Let’s stop
here and
think
about it!
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PROBLEM:



The genomic DNA is 60% G+C
You want a 5000bp fragment
Which enzyme would you try first,
Why?




SmaI (CCCGGG)
EcoRI (GAATTC)
MseI (TTAA)
SacI (GAGCTC)
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Take your time!
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Genomic Libraries
Genomic DNA library
Clone in
vector
Genomic DNA
Digest
DNA fragments
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Choosing a Vector

Usually you select a vector (plasmid, λ,
other) depending on how big you want
your DNA fragments to be & the
capacity of the vector.
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Common vectors used in
library construction
1.
Plasmids

2.
Modified Lambda phage

3.
Up to 10kb inserts
Up to 20kb inserts/40kbp for cosmids
Artificial cloning vectors
BAC- Vectors (bacterial artificial
chromosome)
Up to 100-150kbp inserts

YAC-Vectors (yeast artificial
chromosome)
Up to 500kbp inserts

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Lambda Library
Lodish, et al. Fig 7-12
Plasmid Library
Lodish, et al. Fig 7-1
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Genomic Libraries
Genomic DNA library
Clone in
vector
Genomic DNA
Digest
DNA fragments
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mRNA isolation, purification
Check the RNA integrity
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector
cDNA libraries
1. No cDNA library
was made from
prokaryotic mRNA.
• Prokaryotic mRNA is very unstable
• Genomic libraries of prokaryotes
are easier to make and contain all
the genome sequences.
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cDNA libraries
2.cDNA libraries are very
useful for eukaryotic
gene analysis
•
•
•
•
Condensed protein encoded gene libraries, have
much less junk sequences.
cDNAs have no introns  genes can be expressed in E.
coli directly
Are very useful to identify new genes
Tissue or cell type specific (differential expression of
genes)
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mRNA isolation
Most eukaryotic mRNAs are
polyadenylated at their 3’ ends
5’ cap
AAAAAAAAAAn
• oligo (dT) can be bound to the poly(A) tail and
used to recover the mRNA.
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Check the mRNA integrity
Make sure that the mRNA is not degraded.
Methods:
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
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Cloning the particular mRNAs
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)
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Synthesis of cDNA :
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.
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5’
5’
3’
5’
3’-CCCCCCC
5’-pGGGG-OH
3’-CCCCCCC
5’-pGGGG
3’-CCCCCCC
mRNA
AAAAA-3’
HO-TTTTTP-5’
Reverse transcriptase
Four dNTPs
mRNA
AAAAA-3’
TTTTTP-5’
Terminal transferase
dCTP
mRNA
AAAAA-3’
TTTTTP-5’
cDNA
cDNA
cDNA
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
TTTTTP-5’
Klenow polymerase or reverse
Transcriotase Four dNTPs
-3’
TTTTTP-5’
Duplex cDNA
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5’-pGGGG
3’-CCCCCCC
Duplex cDNA
-3’
TTTTTp-5’
Single strand-specific nuclease
5’-pGGGG
3’-CCC
-3’
TTTTTp-5’
Klenow polymerase
treat with E.coRI methylase
5’-pGGGG
3’-CCCC
Add E.colRI linkers
using T4 DNA ligase
HO-CCGAATTCGGGGGG
3’-GGCTTAAGCCCCCC
-3’
TTTTTp-5’
HO-CCG/AATTCGG-3’
3’-GGCTTAA/GCC-OH
CCGAATTCGG-3’
TTTTTGGCTTAAGCC-OH
E.colRI digestion
5’-pAATTCGGGGGG
3’-CCCCCCC
Fig2.1
CCG-3’
TTTTTGGCTTAAp-5’
Ligate to vector and transfom
Second strand synthesis
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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.
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 )
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Ligation to vector
Any vectors with an EcoRI site would suitable
for cloning the cDNA.
The process :
Dephosphorylate the vector with alkaline
phosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
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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 .
1. Using nucleic acid probe to screen the library
based on hybridization with nucleic acids.
2. Analyze the protein product.
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Screening libraries
Searching the genes of interest in a DNA library
Hybridization to identify the interested DNA or
its RNA product
1.
2.
3.
Radiolabeled probes which is complementary to a region of the
interested gene
Probes:
•
An oligonucleotide derived from the sequence of a protein
product of the gene
•
A DNA fragment/oligo from a related gene of another species
Blotting the DNA or RNA on a membrane
Hybridize the labeled probe with DNA membrane (Southern) or
RNA (Northern) membrane
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Colony and plaque hybridization
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
(Alkali treatment)
Containing Nucleic acid probe)
X-ray
film(radioactively
labeled )
Wash to remove unhybridization probe and visualize
Line up the hybridizated region or
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repeated hybridization
antibody or
enzyme
(modified
nucleotide
labeled
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Transfer to nitrocellulose
or nylon membrane
Keep master
plate
Select positive
from master plate
Denature DNA(NaOH)
Bake onto membrane
Probe with 32p-labled DNA
complementary to
gene of interest
Expose to film
Screening by plaque hybridization
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