Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

1

POLYMERASE CHAIN REACTION (PCR):
PCR, discovered by Kary Mullis in 1985, is an efficient and cost effective molecular tool to
copy or amplify small segments of DNA or RNA
...
Mullis for his invention of the polymerase chain reaction (PCR)
...
It combines principles of complementary
nucleic acid hybridisation with those of nucleic acid replication that are applied repeatedly
through numerous cycles
...
The thermocycler raises and lowers the temperature of the samples in
preprgrammed steps, allowing for denaturation and reannealing of samples with various
reagents
...
It is sensitive and not affected by the quality of DNA like other
techniques such as RFLP (Restriction fragment length polymorphism)
...
During replication, the DNA
molecule unwinds from double stranded DNA to single stranded, and the single stranded DNA
becoming a template for synthesis of a new, complementary strand
...

In vivo replication
Place

where

Feature in PCR

DNA

polymerase attaches to DNA

RNA:DNA

DNA:DNA

strand
Separation of strands of

Helicase

Heat

DNA
Enzyme that elongates new
DNA strands
Primers are made up of

DNA polymerase

Taq polymerase

(Thermolabile)

(Thermostable)

RNA

DNA

2

Steps of PCR:
•

Denaturation: PCR begins with the process of denaturation of a double stranded DNA sample
molecule
...


•

Annealing: In these two synthetic oligonucleotides which are complementary to the target
DNA segment are added to the DNA which has been denatured in the first step
...
It is noted that the synthetic
oligonucleotides which are added to the target DNA should be in high concentration, so as to
ensure that they hybridize with the complementary sequences in the DNA sample
...


•

Extension: The annealed oligonucleotide along with dNTPs and Taq polymerase helps in the
chain elongation process
...
Taq polymerase can
withstand high temperature ranges, as in PCR it can remain active even after being heated
to95◦C and extends the primers which are at temperatures upto 72C
...
After this the temperature of the reaction
is lowered again and another cycle of synthesis of DNA duplex takes place as primer is still
present in excess in the mixture
...

In PCR after each cycle, DNA doubles
...

DENATURATION

EXTENSION

ANNEALING

3

COMPONENTS OF PCR:
• Primers: A primer is a short synthetic oligonucleotide designed to have a
sequence which is the reverse complement of the target region of the
template DNA
...

➢ Inverted repeat sequences should be avoided so as to prevent the
formation of secondary structure in the primer, which may disrupt
the template hybridisation
...

• Taq DNA polymerase: Taq DNA polymerase is a thermostable DNA
polymerase enzyme which can survive high temperature ranges
...

➢ Generally equimolar concentrations of dNTPs are used i
...

➢ Tris-HCl buffer is generally used for PCR as it maintains the pH
between 8
...
8 at room temperature and at 72C pH is 7
...

➢ This reaction mixture of dNTPs and Buffer contains divalent cations
Mg2+ which forms a soluble complex with dNTPs which is essential for
the incorporation of dNTP
...

TYPES OF PCR:
Need of different types of PCR:
•

To reduce contamination

•

In PCR we need to monitor in real time

•

To know how to perform PCR on unknown flanking sequence

•

To perform PCR on RNA sample

1
...
Amplification and simultaneous quantification of target DNA is
done in the same PCR machine using commercially available fluorescence detecting
thermocyclers
...


Working:

5

Detector

Parent
DNA

DNA
polymerase

REAL - TIME PCR

Primer

dNTPs

•

Parent DNA is the DNA sample which is to be amplified
...


•

DNA polymerase (heat resistant), generally Taq polymerase is used
...


Sample preparation

PCR mixture is prepared

Either fluorophore or Taq man probe is added to the PCR mixture

Reaction started

Excitation of fluorescent dye by laser or tungsten lamp

Amplification proceeds

Fluorescent signal is captured after every cycle i
...
in real time

A graph is plotted by the software with cycle number vs
...


➢ SYBER green is green fluorophore dye
...

➢ It is very sensitive to double stranded DNA
...
When it binds with the minor groove
of ds-DNA, it becomes fluorescent
...

Double stranded DNA in PCR binds to DNA binding dye (SYBR green) which leads to
Fluorescence

Intensity of fluorescence at each cycle increases as DNA product increases

Dye binds to all the PCR products including non specific PCR products
Advantages:
➢ Cheap and easy to use
...

Disadvantages:
➢ Non specific in activity
...

•

Taq Man Probes: It recognises specific DNA sequence of interest and probe binds to the
strand
...


7

This method depends on a DNA based probe with a fluorescent reporter at one end and
quencher at opposite end
...

In Taq Man, when the reporter and the quencher are connected to each other, the quencher
reduces the fluorescent signal of the reporter dye as it absorbs the energy via a mechanism
known as fluorescence resonance energy transfer (FRET)
...

Structure of a Taq Man probe:
F

Q

Quencher (Q) is inhibitor of fluorophore (F) as it absorbs light emitted by fluorophore as it
absorbs light emitted by fluorophore
...
The fluorophore is
thereby released from its molecular attachment to the quencher and fluoresces
...


•

It is used to carry out RT-PCR i
...
Real time PCR with many genes
...


•

It is quite expensive
...

Application of Real Time PCR:

•

It is used for the calculation of starting template concentration
...


•

Measuring m-RNA expression
...


•

It has ability to process multiple samples simultaneously
...


2
...

Principle: In this, reverse transcription and PCR amplification can be performed as a twostep process in a single tube or even with two separate reactions
...
The primer used for cDNA synthesis can be either nonsequence specific primers (a mixture of random hexamers or oligo-dT primers) or sequence
specific primers
...


•

Oligo-dT primers are the complementary sequences to the poly-A tail of mRNA molecules and
allow the synthesis of cDNA only from mRNA molecules
...


9

•

They are designed to mRNA molecules of interest which makes reverse transcription a target
specific process
...
But in the process of reverse transcription, cDNA is synthesized from mRNA using
Reverse transcriptase enzyme
...
e
...

Reverse transcriptase enzyme:
Reverse transcriptase is an RNA- dependent DNA polymerase enzyme discovered, by Satoshi
Mizutani and David Baltimore in 1970, in many retroviruses such as HIV and Avian
myeloblastosis virus (AMV)
...
It possesses
three enzymatic activities:
•

RNA-dependent DNA polymerase: It synthesizes a DNA strand complementary to the RNA
...


•

DNA-dependent DNA polymerase: It completes double stranded DNA synthesis
...
But this high error rate of viral reverse transcriptase enzyme provides
selective advantage for their survival in the host system
...
Reverse
transcriptase use an RNA template and a short primer complementary to 3’end of RNA to direct
the synthesis of first strand of cDNA, which can be used directly as a template for the PCR
...


ONE STEP RT-PCR:

mRNA
Reverse transcription

First strand cDNA

PCR cycle 1

PCR cycle 2

AMPLIFICATION

11

ii
...


•

Many clinically important viruses have their genomes composed of RNA, RT-PCR is quite
useful for detecting such viruses like:

➢ Dengue virus
➢ Severe acute respiratory syndrome (SARS)
➢ Human meta-pneumonia virus
➢ Recently it has been used for the detection of Novel Corona Virus in humans
...
Multiplex PCR:
This technology was first used by Chamberlain et al for diagnosis of Duchenne muscular
dystrophy
...

While designing amplification primers for multiplex PCR, several factors must be considered
such as:
•

Primer length: As the technique involves the use of a large numbers of primers, hence it is
required that the designed primer should be of accurate length
...


•

Melting temperature (Tm): Primers which have similar Tm are used
...
The sequences which have high GC (Guanine-Cytosine) content, primer with high Tm
(75◦C – 80◦C) are preferred
...


13

•

Avoiding primer dimer formation: It should be checked that designed primer should not
form primer dimer in the reaction mixture as it leads to reduced yield and non specific
amplification
...


•

Regions with repetitive sequences, called germline, SNPs and regions with high homology
should be avoided as it may affect the efficiency of PCR amplification and create amplification
bias
...
False negatives are often revealed in the multiplex PCR
because each amplicon provides an internal control for other amplified fragments
...


•

Indication of template quality: The quality of template may be dteremined more effectively
in multiplex PCR
...
Nested PCR:
In this PCR type, two sets of primers are used against same target and two successive PCR
reactions occur for the same
...

•

The first set of primer is used for annealing the sequence upstream from the second set of the
primers
...


•

The sec

•

To get information about the size of the insert
...


Procedure:
Extract out cells from the colony in agar plate with a sterile toothpick

Cells are transferred into PCR tubes containing 10µl of 2X PCR buffer
Boil for 5 – 20 minutes and allow it to cool down
(Boiling helps the buffers to extract out DNA from the cells by rupturing their outer
membrane)

Added all the other necessary requirements of the PCR such as dNTPs etc

Performed the PCR

Transferred the PCR product into agarose gel and its analysis is done
(DNA gel analysis)
Advantages:
•

Rapid and cost effective technique
...


•

The setup is quite simple
...


19

•

Restriction digestion for identification of the insert DNA is not required
...

Disadvantages:

•

Although the technique is cost effective, fast and reliable, any mutation cannot be detected
...
Sequencing is required to be
done for the confirmation of the DNA transformation
...

Applications:

•

This technique is widely used for screening of recombinants
...


•

It is widely used for cDNA screening
...
Hotstart PCR:
In conventional PCR we mix all the components like DNA polymerase, MgCl2, PCR buffer,
template DNA, dNTPs, forward and reverse primers in a PCR tube in ice
...
In order to prevent this, we use another technique Hotsart PCR
...

There are three types of hotstart PCR:
•

Manual hotstart PCR: In this PCR we add the reaction components in the reaction tube as in
traditional PCR but we do not add DNA polymerase and MgCl2 in the reaction mixture
...
Then the reaction proceeds and
amplification is performed without any error
...
In order to avoid this we use the
physical barrier hotsart PCR
...
When the temperature
increases the wax melts and it floats up, mixing MgCl2 and DNA polymerase gets mixed with
the main reaction mixture and then the amplification occurs
...
But the wax itself is an external agent which is not suitable in PCR; therefore
recently this method is not in use
...
In this method, DNA polymerase enzyme has the polymerase gets
separated from the monoclonal antibody and binds with DNA template and results in the
amplification
...


➢ Prenatal diagnosis of inherited diseases: It is used for the diagnosis of inherited diseases,
like sickle cell anaemia, beta thalassemia, by using chorionic villus samples or cells from
amniocentesis (covering of foetus)
...

➢ Diagnosis of retroviral infection: PCR (spec

---