PCR BASICS

Kanadi Sumapraja

Departmen Obstetri dan Ginekologi

FKUI-RSCM, Jakarta
 DEFINITION

The polymerase chain reaction is an invitro technique which

allows the amplification of a specific deoxyribonucleic acid
(DNA) region that lies between two regions of known DNA
sequence
PRINCIPLES OF DNA EXTRACTION

• DETERGENT
• ENZYMES
• ALCOHOL
 PRINCIPLES OF DNA EXTRACTION

FIRST STEP: SEPARATING THE CELLS

 PRINCIPLES OF DNA EXTRACTION

SECOND STEP: DISINTEGRATING THE CELL MEMBRANE

 PRINCIPLES OF DNA EXTRACTION
SECOND STEP: DISINTEGRATING THE CELL MEMBRANE
 PRINCIPLES OF DNA EXTRACTION
THIRD STEP: UNMOLDING AND UNFOLDING THE DNA

The enzyme cuts the proteins away from the DNA

 PRINCIPLES OF DNA EXTRACTION
FOURTH STEP: ISOLATING THE DNA

Slowly pour rubbing alcohol (70-95% isopropyl or ethyl alcohol) into the tube
down the side so that it forms a layer on top of the tube (Alcohol is less dense
than water, so it floats on top). DNA will rise into the alcohol layer.
Can this DNA use as sample for gel electrophoresis?

The answer is NO

The DNA that just extracted is genomic, meaning that the entire
collection of DNA from each cell is extracted.
Each cell contains six feet of DNA, but it's only one-millionth of an inch
wide. Therefore, the DNA should be cut with restriction enzymes
Otherwise, genomic DNA is too long and stringy to move through the
pores of the gel; instead, it will end up as a smear.
 RESTRICTION ENZYME

Also known as restriction endonucleases, are enzymes that cut a

DNA molecule at a particular place - looking for a particular
sequence, usually of four to six nucleotides – recognition
sequence - randomly distributed throughout the DNA
Once it finds this particular sequence, it stops and cuts the strands.
RESTRICTION ENZYME
RESTRICTION ENZYME
 Cut vs Uncut DNA

DNA
MARKER Pst I EcoRI UNCUT Pst I EcoRI
 PCR

Double stranded DNA melts

open to become single
stranded DNA and all
enzymatic reaction stops.
This reaction could be done The bases (complementary to the
at the temperature of 94oC. template) are coupled to the primer on the
3' side (the polymerase adds dNTP's from
5' to 3', reading the template from 3' to 5'
side, bases are added complementary to
the template). 72oC is an ideal working
temperature for polymerase

Ionic bonds are constantly formed and broken between the

single stranded primer and the single stranded template. The
more stable bonds may bind a template a bit longer thus
allow DNA polymerase attachment to the little piece of double
stranded DNA (template and primer) and starts copying the
template. Done at the temperature of 54oC
STEPS IN PCR
DNA identification

PCR products should be checked

whether:
-There is a product formed
-The product is of the right size
-Only one band is formed
 RT-PCR
A method which used to make a
huge number copy of DNA from
RNA template using a reverse
transcriptase enzyme
Purpose of this method is to test
whether any specific gene is
turned on (active) or turned off
(inactive).
The first step in RT-PCR uses
reverse transcriptase and a
primer to anneal and extend a
desired mRNA sequence. If the
mRNA is present, the reverse
transcriptase and primer will
anneal to the mRNA sequence
and transcribe a complimentary
strand of DNA – followed by
standard PCR protocol
 Basic of blotting
Molecular searches use one of several forms of complementarity to
identify the macromolecules of interest among a large number of
other molecules.

Complementarity is the sequence-specific or shape-

specific molecular recognition that occurs when two molecules bind
together.
 Hybridization

Complementarity between a probe molecule and a target molecule

can result in the formation of a probe-target complex. This complex
can then be located if the probe molecules are tagged with radioactivity or an
enzyme
 Hybridization

There are two important features of hybridization:

1) Hybridization reactions are specific - the probes will only bind

to targets with complimentary sequence (or, in the case of antibodies, sites
with the correct 3-d shape).

2) Hybridization reactions will occur in the presence of

large quantities of molecules similar but not identical
to the target. That is, a probe can find one molecule of target in a
mixture of zillions of related but non-complementary molecules.
 Hybrids

1) DNA-DNA. A single-stranded DNA (ssDNA) probe

molecule can form a double-stranded, base-paired hybrid with a
ssDNA target if the probe sequence is the reverse complement
of the target sequence.

2) DNA-RNA. A single-stranded DNA (ssDNA) probe

molecule can form a double-stranded, base-paired hybrid with
an RNA (RNA is usually a single-strand) target if the probe
sequence is the reverse complement of the target sequence.

3) Protein-Protein. An antibody probe molecule (antibodies

are proteins) can form a complex with a target protein molecule
if the antibody's antigen-binding site can bind to an epitope
(small antigenic region) on the target protein. In this case, the
hybrid is called an 'antigen-antibody complex' or 'complex' for
short.
Hybrids
 Basic definition

Southern Blot
DNA cut with restriction enzymes - probed with radioactive DNA.

Northern Blot
RNA - probed with radioactive DNA or RNA.

Western Blot
Protein - probed with radioactive or enzymatically-tagged antibodies.
 Basic

In the case of Southern, Northern, and Western blots, the initial separation
of molecules is done on the basis of molecular weight

- Gel electrophoresis
- Transfer to Solid Support
- Blocking
- Preparing the Probe
- Hybridization
- Washing
- Detection of Probe-Target Hybrids
 Gel electrophoresis

The pH and other buffer conditions are arranged so that the molecules
being separated carry a net (-) charge so that they will me moved by
the electric field from left to right. As they move through the gel, the
larger molecules will be held up as they try to pass through the pores
of the gel, while the smaller molecules will be impeded less and move
faster. This results in a separation by size, with the larger molecules
nearer the well and the smaller molecules farther away.
Gel electrophoresis
 Transfer to solid support

After the DNA, RNA, or protein has been separated by molecular

weight, it must be transferred to a solid support before hybridization.
(Hybridization does not work well in a gel.) This transfer process is
called blotting and is why these hybridization techniques are called
blots. Usually, the solid support is a sheet of nitrocellulose paper
 Transfer to solid support
The DNA, RNA, or protein can be transferred to nitrocellulose in one of two ways:
1) Electrophoresis, which takes advantage of the molecules' negative charge:

2) Capillary blotting, where the molecules are transferred in a flow of buffer from
wet filter paper to dry filter paper:
 Blocking

The filters are soaked in a blocking solution which contains a high

concentration of DNA, RNA, or protein. This coats the filter and
prevents the probe from sticking to the filter itself.
Preparing the probe
Hybridization-washing-detection