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Genomic and cDNA libraries
DNA Libraries
…collections of cloned DNA fragments,
– genomic,
– cDNA (coding sequences).
Genomic Library Construction
• Cloning all fragments of an organisms genome
= genomic library
– Each fragment in a vector transformed into a
bacterial cell = Book
– The collection of all of the clones (thousands) is
the genomic library
– requirement: *random
* bigger is better
Genomic Library Construction
DNA fractioning
• DNA shearing
• to ensure that DNA fragments are of size
suitable for cloning
– Digest with enzyme with 4bp recognition site
– One site every 256bp (1/4 x 1/4 x 1/4 x 1/4)
– Do a partial digestion (not all sites cleaved)
– Ligate fragments to vector
– Transform into E. coli
Genomic Sequences and Coverage
N = ln(1 - P)
ln(1 - f)
N = number of clones
P = probability of recovering a sequence,
f = fraction of the genome of each clone
f = genome lenght
average lenght of insert
Probability
0.99
0.95
0.9
0.8
Insert lenght
15kb
40kb
860000
560000
430000
300000
320000
210000
160000
115000
Results for the human genome
Making a genomic DNA library.
Production of overlapping restriction fragments by partial digestion of human
genomic DNA with Sau3A. This restriction endonuclease recognizes the 4-bp sequence GATC and
produces fragments with single-stranded sticky ends with this sequence on the 5 end of each strand. A
hypothetical region of human genomic DNA showing the Sau3A recognition sites (red) is shown at the
top. Partial digestion of this region of DNA would yield a variety of overlapping fragments (blue) ≈20 kb
long. Use of such overlapping fragments increases the probability that all sequences in the genomic DNA
will be represented in a l library.
Partial Digest
Cosmid Cloning
Cloning in Cosmids
Production of overlapping restriction fragments by partial digestion of human
genomic DNA with Sau3A.
CHROMOSOME WALKING
CHROMOSOME WALKING
Chromosome ‘Walking’
DNA Libraries
…collections of cloned DNA fragments,
– genomic,
– cDNA (coding sequences).
Cloning euykaryotic genes in prkaryotes require special "tricks" because eukaryotic
genes have introns which are removed in the nucleus of eukaryotic cells prior to
translation
Introns can account for more than 90% of the length of a eukaryotic gene. It is hard to clone
very long DNA segments. In addition, intron-containing eukaryotic genes cannot be expressed
in a bacterial host because prokaryotes lack splicing apparatus.
To overcome these problems, instead of directly cloning a gene, one can clone cDNA, a DNA
copy of gene mRNA.
An enzyme, reverse transcriptase, is used to produce cDNA
cDNA
…DNA synthesized from an mRNA template
with the enzyme reverse transcriptase.
Reverse Transcriptase
1. RNA dependent, DNA synthesis.
2. RNA Degradation.
3. DNA dependent, DNA Synthesis.
Basically two types:
- AMV (aviary myeloblastosis)
- MMLV (Moloney Murine Leukemia Virus)
Error Rate: 1 in 20,000 nucleotides.
Isolation of mRNA by oligo-dT affinity chromatography.
cDNA Construction
in vitro
RNA degradation by alkali
DNA pol and second strand synthesis
Cleavege of hairpin by Nuclease S1
Terminal transferase and ligation into vector
cDNA
Strategy to
synthetize
double strand cDNA
B) cDNA Synthesis
mRNA
AAAAAn
Anneal oligo dT primer
primed mRNA
AAAAAn
TTTTT
Reverse Transcriptase
and dNTPs
mRNA/cDNA
hybrid
AAAAAn
TTTTT
Gubler Hoffman cDNA Synthesis
mRNA/cDNA
hybrid
AAAAAn
TTTTT
RNase H
nicked RNA
AAAAAn
TTTTT
DNA Pol I
nicked RNA used
as primers by Pol
AAAAA
TTTTT
Gubler Hoffman cDNA Synthesis
2nd strand cDNA
in pieces
AAAAA
TTTTT
E. coli DNA
Ligase
AAAAA
ds cDNA
TTTTT
Clone into vector
cDNA Library
cDNA Libraries
…provide a ‘snap-shot’ of the genes expressed in
a particular cell, at a particular time, or under
specific condition,
…however, do not provide regulatory sequences.
cDNA Libraries Limits:
…using a oligo dT, library has 3’-rich sequences
Using random examers it’s possible overcome
this drawbacks, but the average length of
sequences is reduced.
…hard to isolate long and full-lenght transcripts.
CAPture method:
AAA
Full cDNA
incomplete cDNA
AAA
TTT
AAA
TTT
Rnase A
AAA
TTT
AAA
TTT
Chromatography for elF-4E
AAA
TTT
Elution of full-lenght cDNA
BAP= Alkaline phosphatase
TAP= acid pyrophosphatase
5’ - RACE Rapid Amplification Complementary Ends
mRNA 7-mG
AAAAA-3’
Sintesi primo filamento cDNA
Trascrittasi inversa
+ GSP1
7-mG
GSP1
Aggiunta coda omopolimera
al primo filamento cDNA
GSP1 5’
P
TTTT
P
TTTTT
3’
TTTTT
GSP1’ 3’
3’
P
TTTTT
P’
AAAAA
GSP1 5’
complementarietà
al primer GSP1
GSP1 5’
I° gruppo di amplificazioni
5’
P
GSP2
P
TTTTT
3’-AAAAAAAAA
5’
Trasferasi terminale
+ dATP
3’-AAAAAAAAA
Sintesi secondo filamento cDNA
AAAAA-3’
II° gruppo di amplificazioni
GSP1’ 3’
GSP1 5’
complementarietà
al primer P(dT)
5’
P
TTTTT
GSP1’ 3’
3’
P’
AAAAA
GSP1 5’
5’
P
TTTTT
GSP2’ 3’
3’
P’
AAAAA
GSP2 5’
Prodotto PCR finale
3’ - RACE Rapid Amplification Complementary Ends
mRNA
7-mG
AAAAAAAA-3’
Sintesi primo filamento cDNA
Trascrittasi inversa
+ P(dT)
7-mG
AAAAAAAA-3’
TTTTT
Sintesi secondo filamento cDNA
GSP1
P
complementarietà
al primer P(dT)
GSP1
3’
TTTTT
complementarietà
al primer GSP1
P
5’
I° gruppo di amplificazioni
5’ GSP1
AAAAA
P’
3’
3’ GSP1’
TTTTT
P
5’
II° gruppo di amplificazioni
3’ GSP1’
GSP2
5’ GSP1
TTTTT
P
TTTTT
P
AAAAA
P’
5’
3’
5’ GSP2
AAAAA
P’
3’
3’ GSP2’
TTTTT
P
5’
Prodotto PCR finale
Identification of a specific clone from a library by membrane
hybridization to a radiolabeled probe
Source of the DNA Probe?
• Probe DNA must have complementarity with
target DNA
• Two sources
– Related organism - heterologous probe
– Reverse translate protein sequence
DNA Probe
• Probe from Related Organism
– sequences are related evolutionarily
– not identical but similar enough
– Heterologous Probe
– Use yeast gene to isolate Human gene
DNA Probe
• Reverse translate protein sequence
– Use knowledge of Genetic Code to obtain DNA
sequence(s) based on protein seq.
– MET = AUG in RNA
ATG in DNA (or 5’ CAT)
Genetic
Code
Table
Reverse Translation of Protein Seq.
MET-TRP-TYR-GLN-PHE-CYS-LYS-PRO
ATG-TGG-TAT-CAA-TTT-TGT-AGA-CCN
C
G C
C
G
32 Different oligonucleotides for this peptide
sequence (due to degeneracy of code)
Radiolabeling of an oligonucleotide at the 5 and with phosphorus-32. The three phosphate
groups in ATP are designated the a, b, and g phosphates in order of their position away from the
ribose ring of adenosine (Ad). ATP containing the radioactive isotope 32P in the g-phosphate
position is called [g-32P]ATP. Kinase is the general term for enzymes that transfer the gphosphate of ATP to specific substrates. Polynucleotide kinase can transfer the 32P-labeled g
phosphate of [g-32P]ATP to the 5 end of a polynucleotide chain (either DNA or RNA). This
reaction is commonly used to radiolabel synthetic oligonucleotides.
Labelling of PCR products using a radioactive
dNTP!
Designing oligonucleotide probes based on protein sequence. The determined sequences then are analyzed to
identify the 6- or 7-aa region that can be encoded by the smallest number of possible DNA sequences. Because of the
degeneracy of the genetic code, the 12-aa sequence (light green) shown here theoretically could be encoded by any of the
DNA triplets below it, with the possible alternative bases at the same position indicated. For example, Phe-1 is encoded by
TTT or TTC; Leu-2 is encoded by one of six possible triplets (CTT, CTC, CTA, CTG, TTA, or TTG). The region with the least
degeneracy for a sequence of 20 bases (20-mer) is indicated by the red bracket. There are 48 possible DNA sequences in this
20-base region that could encode the peptide sequence 3 9. Since the actual sequence of the gene is unknown, a degenerate
20-mer probe consisting of a mixture of all the possible 20-base oligonucleotides is prepared. If a cDNA or genomic library is
screened with this degenerate probe, the one oligonucleotide that is perfectly complementary to the actual coding sequence
(blue) will hybridize to it.
Colony PCR
2. Si mette su una reazione di PCR per ogni
clone che si vuole analizzare, risospendendo
nella mix di PCR una parte della colonia.Si
utilizza una coppia di primers specifica per
l’inserto clonato
1. Si piastra, come al solito, una
trasformazione. I cloni trasformanti
possono contenere il solo vettore o il
vettore più l’inserto
M
1
2
3
4
5
_
+
I cloni 1,2,3 e 4 contengono l’inserto. Il clone 4 contiene solo il vettore. “-” e “+” sono controlli negativo e positivo
Immunological Screening
• Antibodies to the protein encoded by the
desired gene can be used to screen a
library
Immunological Screen
master plate
matrix
1
6
transfer
cells
cells
protein
2
lyse cells
bind protein
subculture
cells from
master plate
3
positive
signal
5
4
Add substrate
Add 2°Ab-Enzyme
wash
Add 1°Ab;
wash
Immunological Screen of Library
substrate
detectable
product
reporter enzyme
2° Ab
1° Ab
target protein
matrix
o---- Cloning strategies ----o
I.
II.
Making DNA “libraries” (from genomic DNA, mRNA
“transcriptome”)
Screening to identify a specific clone (the needle in
the haystack)
-- by the sequence of the clone
-- by the structure or function of the expressed
product of the clone
Course reading: #28
(and 29)
Overview of strategies for cloning genes
1)
2)
3)
Get DNA
Ligate to vector
Transform or transfect
Look for the gene…
4)
1) Get DNA
Genomic DNA
RNA
Ligate to vector: how to make this reaction favorable?
This yields a “library”, a representative set of all the pieces of DNA that make up a
genome (or all the cDNAs that correspond to the “transcriptome”)
cDNAs from different tissues reflect the different RNA populations that you find in
distinct cell types:
Hence “liver” vs. “brain” vs. “heart” cDNA libraries
There are lots of ways to identify a particular gene…
Overview of strategies for cloning genes