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 5terminus 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:

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 P H 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/.