Transcript PRIMER EXTENSION, AND NUCLEASE SI MAPPING.

PRIMER EXTENSION AND
NUCLEASE S1 MAPPING.
BIOCHEMISTRY SEMINAR.
ALICE MUMBEY-WAFULA
FEB, 20TH 2004.
PRIMER EXTENSION.
• This is a method which employs the enzyme
Reverse Transcriptase,which is also known as
RNA-dependent DNA polymerase.
• Reverse Transcriptase was discovered in 1970 by
Howard Temin and David Baltimore
• It is obtained mainly from tumour causing
viruses, like Avian Myeloblastosis Virus.
• It catalyses the synthesis of cDNA using an RNA
as a template.
Uses:
• Primer extension is used to measure the
amount of a given RNA and to map the 5’
end of that RNA.
• The creation of cDNA libraries from
mRNA.
• Detection of transcripts produced during in
vitro transcription.
• Mapping of replication initiation point.
Principle of Analysis
• Like all other polymerases, this enzyme requires
both a template and a primer to be extended.
• In this protocol, the primer extension is used to
map the 5’terminus of an RNA and to quantitate it.
• A DNA end-labeled primer is annealed
(hybridized) to the RNA template and extended by
reverse transcriptase using unlabeled
deoxyribonucleotides.
• The resultant cDNA formed is analyzed on
a sequencing gel.
• The length of the extended primer maps the
position of the 5’ end of the RNA, and the
yield of primer extension product reflects
the abundance of the RNA.
Materials:
Annealing mix:
- RNA
- Radio-labeled oligonucleotides (primer with
defined sequence).
- 0.1 M Tris.Cl PH 8.3
- 0.5M Mg.Cl2
- RNase fre water.
Materials:
Synthesis mix;
AMV reverse transcriptase
1 M Tris.Cl PH 8.3
0.5 M Mg.Cl2
Actinomycin
4 dNTP mix
RNAse free water
Other materials:
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RNAse reaction mix.
3 M sodium acetate
Phenol/chloroform/isoamyl alcohol.
100% and 70% Ethanol.
Loading dye
9% polyacrylamide.
Method:
1- Annealing RNA and primer;
RNA, dissolved in RNAse free water is
mixed with annealing buffer containing
one-end radio-labeled primer in eppendorf
tubes. The tubes are incubated for 1 min. in
a boiling water bath.
Method
2- Primer extension reaction;
The synthesis mix is then added into the tubes and
incubated for 1 hr at 42oC.
3- Degradation of RNA;
The RNAse reaction mix is then added to each
primer extension tube, incubated for 15 mins at
37oC. Rnase degrades the template RNA, leaving
a cleanly labeled single-stranded DNA product.
The reactions catalyzed by reverse
transcriptase.
The X-ray
Structure and
Ribbon
diagram of
HIV-1 reverse
Transcriptase.
Method, ctd:
• DNA is then extracted from the product
using sodium acetate,
phenol,/chloroform/isoamyl alcohol. The
aqueous layer which contains DNA is
transferred into a fresh tube.
Method (cont.)
• DNA is precipitated in 100% ethanol, the pellet is
washed in 70% ethanol and air dried for 5 to 10
min. The pellet is resuspended in loading gel,
heated for 5 min in 65oC water bath.
• The samples are loaded in 9% polyacrylamide/7
M urea gel. The length of the cDNA gives the
complete map of the RNA up to 5’ end.
• Markers of known sizes and length are loaded
along side the samples.
The sample is
ran together with
markers of
known sizes ,
which helps in
the determination
of the length and
size.
Polyacrylamide gel electrophoresis, samples
1-4 and markers A &C
Advantage:
No probe is used, it is simple and straight
forward.
Disadvantage:
Reverse transcriptase sometimes encounters
regions of RNA that cause it to pause or
terminate synthesis.
This mostly occurs in G-C rich stretches.
It results in bands of intermediate size and
confuse the interpretation, and reduce the
yield of fully extended primer.
NUCLEASE S1 MAPPING.
Background:
• Is a single strand- specific endonuclease.
• It is isolated from Aspergillus oryzae
amylase.
• It digests both single-stranded DNA &
RNA.
• Is a metalloenzyme with zinc bound on to
its active site.
• Is thermostable & resistant to
denaturants/high salt concentration.
• It works best in an acidic PH.
Uses:
• Mapping 5’ & 3’ ends of a transcript using
labeled probe.
• Quantitating the level of a particular RNA.
• Mapping locations of introns.
• Analyzing the structure of DNA:RNA
hybrids.
Uses:
• Removing single-stranded overhangs from
DNA fragments to produce blunt ends.
• Opening the hairpin loops generated during
synthesis of dsDNA.
Principle of Analysis:
• Hybridization of labeled DNA probe
fragment to cellular RNA.
• Digestion by nuclease of all the singlestranded regions, RNA:DNA hybrid is
resistant to this cleavage.
• Product is ran on a polyacrylamide gel.
• The labeled DNA fragment then reflects the
amount and size of the RNA in the hybrid
that is homologous to the DNA probe.
Materials:
Hybridization buffer;
• 3 M Nacl2
• 10 mM EDTA
• 380 mM HEPS PH 7.0.
(cont.)
Hybridization mix.
• RNA
• Labeled oligonucleotide probe(DNA)
• Hybridization buffer
• Triton X-100
• RNase free water.
S1 Digestion mix.
• 0.33 M NaCl2
• 60 mM NaoAc PH 4.6
• 2.2mM Znso4
• 0.01% Triton X-100
• 20 units S1 nuclease
• RNase free water
Stop mix:
• 2.5 M NaoAc
• 40 mM EDTA
• 3ug tRNA.
Method:
• Hybridization of RNA & 32P labeled DNA probe.
All tubes must be RNase free.
• Hybridization mix is put in a microcentrifuge
tube,placed in a thermal cycler and heated for 3
min at 94oC (denaturing).
• Temperature is reduced to 55oC and sample
incubated overnight ( at least 10 hrs.) for complete
hybridization. Temp. can be adjusted according to
application.
S1 nuclease Digestion:
S1 nuclease Digestion.
• Hybridization tubes are removed and span
quickly, content transferred to 1.5 ml
eppendorf tubes.
• S1 nuclease digestion mix is added, mixed
well and span.
• Tubes are incubated at 37oC for 30 min.
Stop reaction:
• The reaction is stopped by adding stop mix.
• Samples are precipitated using 100% ethanol.
• Pellet is washed using 70% ethanol and dried for
5 min in a speedvac-evaporator.
• Pellet is resuspended in TE (Tris & EDTA) and
loading dye(bromophenol blue).
• Tubes are boiled for 3 min and placed on ice.
Electrophoresis:
• Samples are loaded on a 6.8% acrylamide
gel and separated by electrophoresis.
• Labeled markers are loaded along with the
samples.
• The gel is dried and exposed to X-ray film.
• Results are read directly from the
autoradiogram.
An example
Of an autoradiograph,
Showing bands
with 32P.
Disadvantage of analysis.
• Depurination may occur due to low PH at
which nuclease works. This limits its
applications.
• If high concentration is used, a break can be
created in the DNA duplex.
• Use of labeled probe makes it a
complicated procedure.
References:
• Voet D; Voet J. Biochemistry
• A. Kornberg and Baker. DNA replication.
• Current protocols in molecular biology.
Wiley and sons.
• http://www.mrw2.interscience.wiley.com/cp
online/tserver.
• http://www.fhcrc.org/labs/hahn/methods/mo
l-bio-meth/.