POLYMERASE CHAIN REACTION METHOD
Polymerase Chain Reaction:
PCR stands for the
Polymerase Chain Reaction. It is a
patented procedure developed by Kary Mullis in 1987, which can enzymatically
amplify minute quantities of DNA to large number of copies. By this method, a
single copy of a nucleic acid that is often difficult to detect by standard
hybridization methods, is multiplied to ≥107 copies in a short
period.
Mechanism
of PCR:
In 1985-86 a major
development occurred at Cetus Corporation, USA where researchers develop an
in vitro method for the amplification of DNA fragment. This method is known as
polymerase chain reaction (PCR). The technique becomes important with the discovery
of Taq DNA Polymerase by Kary Mullis 1987.Now a days Polymerase chain reaction
is a widely used technique. It provides a simple method for exponential
amplification for specific DNA sequence in vitro.
(a) Principle:The process of amplifying the target DNA begins with designing a pair of primers
complementary to the two regions spanning the target on either strand.The two
strands of the target DNA are separated (denaturation) by heating and the
primers are applied.The primers bind to their targets on either strands.These
primers are then elongated by DNA polymerase enzyme complimentary to the
nucleotides on the target on both strands. Thus, each single strand of the
target gets its complimentary strand, resulting in production of two target DNA
molecules from a single DNA molecule. This process is repeated several times in
order to obtain larger number of copies. The amplified target DNA segments are
called amplicons.
(b) Requirement: All the steps of PCR are performed on
the reaction mixture consisting of target DNA, primer pairs, thermostable DNA
polymerase, deoxynucleotides (dATP, dTTP, dGTP & dCTP), buffer and Mg salt
in the same test tube.This process require following
materials.
(i) Template DNA or Target DNA: DNA, which is to be amplified is known
as target DNA.
(ii)Primer: Primers are short, single stranded
oligonucleotide DNA that are 20-30 nucleotides in length. These are chemically
synthesized and are always designed in pairs, complementary to opposite strands
of the target. Two primers specific to 3' ends of concern DNA
segment are required in PCR. Primers are so designed that they hybridize with the target 50-3000
nucleotides apart from each other. In other words, primers bind to the region
flanking target sequences that are 50-3000 nucleotides long.Each primer must be
devoid of palindromic sequence that can
give rise to stable intrastrand structuresthat limit primer annealingto the
template strand.
(iii) Amplification buffer: The standard buffer for PCR reaction contains
KCl, Tris-Cl, 1.5mM MgCl2.To maintain the pH presence of divalent magnesium cation is
critical.
(iv)dNTP:Four types of deoxynucleotide triphosphates (dNTPs) are used
at saturating concentration of 200
(v) DNA polymerase: A heat stable DNA polymerase, Taq i.e. Thermus
aquaticus DNA polymerase Pfu i.e. Pyrococcus furiosus. This type of
DNA polymerase is heat resistant and hence it is not necessary to freshly add
this enzyme for each cycle of PCR.
(c)Steps of PCR: Action of PCR
involves several cycles of amplification however each cycle has 3 steps-
denaturation, primer annealing and primer extension (polymerization).
(i) Denaturation: In this step, the
template is denatured by raising the temperature to a point where the bonds
holding double stranded structure of DNA are disrupted. The denaturation
temperature varies between 90-97°c. (usually 94°c). template with high
temperature in denaturation of the strand. For efficiency reaction mixture is
heated to 94°c for 2 minutes before polymerase, Taq DNA polymerase.
(ii) Primer annealing:Short single stranded
DNA molecules are called primers. They are generally between 16-40 basepairs in
length with exact match of template sequence is not defined a mixture of primer
is used. The perfect matching of primer with the template DNA require correct
annealing temperature. The perfect matching primer generally require annealing
temperature of 35-60°c. whereas the mixture of primer with some random
nucleotide (degenerate primer) require 45-55°c for annealing. Generally primers
are added in excess so that the anneal with the template can anneal with each
other. The duration of annealing step is usually 1minute.
(iii) Primer extension and chain elongation:This
chain extension process carriedout in 72°c. The basic steps of primer extension
are as follows-
a) Taq DNA
polymerase and deoxyribonucleoside triphosphates are added to the mixture to
initiate the synthesis of two new chains complementary to the original DNA chains.
b)
The DNA polymerase adds nucleotides to the 3' hydroxyl end of the primer and
strand growth extends.
The reaction of chain
elongation is exactly similar to the DNA replication that occurs in vivo.
Repeatation of
steps:After
one cycle of replication, the reaction mixture is heated again to denature the
DNA strands. Each DNA strand again binds a complementary primer and the cycle
of chain extension is repeated. Typically 20-30 cycles are run during DNA
amplification. The duration of each elongation is usually 2 minutes. Each newly
synthesized polynucleotide can act a template for the successive cycles.Each
cycle of PCR takesabout 3 to 5minutes.
For the second
cycle of PCR , the , the DNA strands (original+newly synthesized long
templates) are denatured, annealed with primers and subjected to DNA
synthesis.At the end of the second round long templates and short templates are
formed.In the third cycle of PCR, the original DNA strands along with long and
short templates are the starting materials. The technique of denaturation,
renaturation and synthesis are repeated.This procedure is repeated again and
again for each cycle.It is estimated that at the end of 32 cycles of PCR about
a million foldtarget DNA is synthesized. The short templates possessing
precisely the target DNA as double stranded DNA accumulates.
Role of PCR in modern biology:
The
advent of PCR have a continuous and tremendous impact on the molecular biology.
The applications of PCR are too many to be listed here some of them are
selectively and very briefly described.
(i)
Diagnosis inherited disease: The specificity and sensitivity of PCR is highly useful for
the diagnosis of various diseases...........
(a) PCR employed
in the prenatal diagnosis of inherited diseases by using chorionic villus
samples or cells from amniocentesis.Thus diseases like sickle-cell anemia ,
β-thalassemia and Phenyl ketonurea can be detected by PCR.
(b)PCR from cDNA
is a valuable tool for diagnosis and monitororing of retroviral infections; eg
HIV infection. It is also used in diagnosis of virus mediated cancer.
(ii) PCR in sex determination of embryo:Sex
of human, life stock and higher plant embryo fertilized in vitro can be
determined by PCR by using primers and DNA probes specific for DNA probes.
Further this technique is also useful to detect sex linked disorders in
fertilized embryos.
(iii)DNA sequencing:As the PCR technique is
much simpler and quicker to amplify the DNA it is conveniently used for
sequencing. It is also employed in various molecular marker technique like
RAPD, RFLP, AFLP etc.
(iv)
Detection of Pathogen:There are
several pathogens that grow slowly. Therefore, their cells are found laws in
number in the infected cell or tissue. It is difficult to culture them on
artificial medium. Hence for their diagnosis PCR based assays have been
developed.
(v)PCR in gene manipulation and expression
studies: The advantage with PCR is that the primers need not have
complementary sequences for the target DNA. Therefore the sequences of
nucleotides in a piece of gene can be manipulated and amplified by PCR. By side
directed mutagenesis coding sequence can be altered to synthesize protein of
interest.
(vi)
Paleontological diagnosis:PCR
technique has been used to clone the DNA fragments from the mummified remains
of humans and extinct animals or plants. DNA from buried sample have been
amplified and uswed to trace the evolution biological migration etc.
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