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PCR Assignment

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89 views3 pages

PCR Assignment

Uploaded by

surbhimakwana3
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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25th November,2020

PCR

PRINCIPLES:-
• The basic PCR principle is simple. As thename implies, it is a chain reaction: OneDNA
molecule is used to produce twocopies, then four, then eight and so forth.
• Thiscontinuous doubling is accomplishedby specific proteins known aspolymerases,
enzymes that are able tostring together individual DNA buildingblocks to form long
molecular strands.
• Todo their job polymerases require a supplyof DNA building blocks, i.e.
thenucleotides consisting of the four basesadenine (A), thymine (T), cytosine (C)and
guanine (G).
• They also need a smallfragment of DNA, known as the primer,to which they attach
the building blocksas well as a longer DNA molecule toserve as a template for
constructing the new strand.
• If these three ingredients aresupplied, the enzymes will construct exactcopies of the
templates.
THEORY:-

• The polymerase chain reaction (PCR) is a scientific technique in molecular biology


to
• Amplify a single or a few copies of a piece of DNA across several orders of
magnitude,
• Generating thousands to millions of copies of a particular DNA sequence.
Polymerase Chain Reactionwas developed in 1984 by the Americanbiochemist, Kary
Mullis. Mullis receivedthe Nobel Prize and the Japan Prize fordeveloping PCR in
1993principle.

Each synthesis cycle is composed of three step.

1) Denaturation.
During the denaturation step, the reaction cocktail is exposedto high temperature, usually
95oC. This high temperature will denature theDNA—meaning the two complementary
strands of the DNA molecule unravel,exposing the nucleotide bases. The high temperature
of the denaturing stephas the added advantage of denaturing proteins and disrupting cells
so youdon’t have to always start with purified DNA as your amplification template,you can
often times amplify DNA directly from cell lysates—or even wholecells.
2) Primer Annealing.
During the second step of each cycle, the temperature islowered to an annealing
temperatureallow annealingof the primers to their complementary targets on the DNA
template (one foreach DNA strand). These are designed to flank the desired target region
ofyour DNA template and serve as the starting points for DNA synthesis by
theTaqpolymerase.

3) Extension.
The reaction cocktail is now brought to the optimum reactiontemperature for Taq
polymerase (68 to 72oC). During this step, the Taqwillbind to each DNA strand and “extend”
from the priming sites (synthesize acomplementary strand of the targeted
APPLICATION OF PCR:-

• Amplification of small amounts of DNA for further analysis by DNA fingerprinting.


• The analysis of ancient DNA from fossils.
• Mapping the human (and other species) genome.
• The isolation of a particular gene of interest from a tissue sample.
• Generation of probes: large amount of probes can be synthesized by this technique.
• Production of DNA for sequencing: Target DNA in clone is amplified using
appropriate primers and then itssequence determined. Helpful in conditions where
amount of DNA is small.
• Analysis of mutations: Deletions and insertions in a gene can be detected by
differences in size of amplifiedproduct.
• Diagnosis of monogenic diseases (single gene disorders): For pre-natal diagnosis,
PCR is used to amplifyDNA from foetal cells obtained from amniotic fluid. PCR has
also proved very important in carrier testing.
• Detection of microorganisms: Especially of organisms and viruses that are difficult
to culture or take longtime to culture or dangerous to culture.
• The PCR has even made it possible to analyze DNA from microscope slides of tissue
preserved yearsbefore.
• Detection of microbial genes responsible for some aspect of pathogenesis or
antibiotic resistance.
• Crucial forensic evidence may often be present in very small quantities, e.g. one
human hair, body fluidstain (blood, saliva, semen). PCR can generate sufficient DNA
from a single cell.

Different between the PCR and RT-PCR :-

2
1 .PCR is a technique to amplify a segment of 1. RT-PCR is a variant of PCR used in the
DNA, generating millions of copies of a DNA detection of gene expression in molecule
sequence biology

2 .Denaturation, annealing, and extension are 2. Reverse transcription is followed by PCR


the three steps
3. A single-stranded RNA molecule is the
3. A double-stranded DNA molecule serves as template for the reverse transcription; a single-
the template strand DNA molecule is the template for the
PCR
4. DNADNA polymerase is used as the enzyme
4. Reverse transcriptase and DNA polymerase
5. Forward and reverse primers are used are used as enzymes

6 .Comparatively less sensitive 5. Reverse primer is used for reverse


transcription
7. Used in functional analysis of genes,
diagnosis, and monitoring of hereditary 6. More sensitve method
diseases, DNA cloning, DNA sequencing, and
ancient DNA amplification 7. Used in the detection of gene expression

LIMITATIONS OF PCR:-

• PCR is an extremely sensitive technique but is prone to contamination from


extraneous DNA,leading to false positive results.
• Another potential problem is due to cross-contamination between samples. It is
forthis reason that sample preparation, running PCR and post-amplification
detection must be carried out in separaterooms.
• Concentration of Mg is very crucial as low Mg2+ leads to low yields (or no yield) and
high Mg2+ leads toaccumulation of nonspecific products. Non-specific binding of
primers and primer-primer dimmerformation are otherpossible reasons for
unexpected results.
• Reagents and equipment are costly, hence can’t be afforded by smalllaboratories.

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