ABHISHEK S3 APA PCR advanced pharmaceutical analysis
1. SUBJECT : APA
KARNATAKA COLLEGE OF PHARMACY
TOPIC : POLYMERASE CHAIN REACTION
SUBMITTED BY: SUBMITTED TO:
ABHISHEK DR. C. SREEDHAR
M. PHARM 1ST SEMESTER PROF. AND HOD
DEPT. OF PHARMACEUTICAL ANALYSIS DEPT. OF PHARMACEUTICAL ANALYSIS
KARNATAKA COLLEGE OF PHARMACY KARNATAKA COLLEGE OF PHARMCY
BANGALURU BANGALURU
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3. INTRODUCTION
• Polymerase Chain Reaction (PCR) is a powerful method for amplifying segments of
DNA, distinct from cloning and propagation within the host cell.
• This procedure is carried out entirely biochemically, that is, in vitro.
• PCR was invented by Kary Mullis in 1983.
• He shared the Nobel Prize in chemistry with Michael Smith in 1993.
• PCR has made it possible to generate millions of copies of a small segment of DNA.
• This tool is commonly used in the molecular biology and biotechnology labs.
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4. Principle of PCR
PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from
deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase adds
nucleotides to the 3` end of a custom-designed oligonucleotide when it is annealed to a
longer template DNA. Thus, if a synthetic oligonucleotide is annealed to a single-stranded
template that contains a region complementary to the oligonucleotide, DNA polymerase
can use the oligonucleotide as a primer and elongate its 3` end to generate an extended
region of double stranded DNA.
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5. Components Of PCR
1. DNA Template:– The DNA of interest from the sample.
2. DNA Polymerase:– Taq Polymerase is used. It is thermostable and does not denature at
very high temperatures.
3. Oligonucleotide Primers:- These are the short stretches of single-stranded DNA
complementary to the 3’ ends of sense and anti-sense strands.
4. Deoxyribonucleotide triphosphate:– These provide energy for polymerization and are
the building blocks for the synthesis of DNA. These are single units of bases.
5. Buffer System:– Magnesium and Potassium provide optimum conditions for DNA
denaturation and renaturation. It is also important for fidelity, polymerase activity, and
stability.
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7. PCR Steps/Procedure:-
The PCR involves three major cyclic reactions:
1. Denaturation
• Denaturation occurs when the reaction mixture is heated to 94℃ for
about 0.5 to 2 minutes. This breaks the hydrogen bonds between the two
strands of DNA and converts it into a single-stranded DNA.
• The single strands now act as a template to produce new strands of DNA.
The temperature should be provided for a longer time to ensure the
separation of the two strands.
2. Annealing
• The reaction temperature is lowered to 54-60℃ for around 20-40
seconds. Here, the primers bind to their complementary sequences on the
template DNA.
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8. • Primers are single-strand sequences of DNA or RNA around 20 to 30 bases in length.
• They serve as the starting point for the synthesis of DNA.
• The two separated strands run in the opposite direction and consequently there are two
primers- a forward primer and a reverse primer.
3. Elongation
• At this step, the temperature is raised to 72-80℃. The bases are added to the 3’ end of
the primer by the Taq polymerase enzyme.
• This elongates the DNA in the 5’ to 3’ direction. The DNA polymerase adds about
1000bp/minute under optimum conditions.
• Taq Polymerase can tolerate very high temperatures. It attaches to the primer and adds
DNA bases to the single strand. As a result, a double-stranded DNA molecule is
obtained.
• These three steps are repeated 20-40 times in order to obtain several sequences of DNA
of interest in a very short time period.
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10. Types of PCR
1.Real-time PCR
2.Nested PCR
3.Multiplex PCR
4.Quantitative real time PCR (Q-RT PCR)
5.Reverse Transcriptase PCR (RT-PCR)
6.Long-range PCR
7.Single-cell PCR
8.Fast-cycling PCR
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11. PCR Studies for gene regulations:-
Gene regulation is the process used to control the timing, location
and amount in which genes are expressed
Real-time polymerase chain reaction (real-time PCR) is commonly
used to measure gene expression. It is more sensitive than
microarrays in detecting small changes in expression
The PCR in combination with prior reverse transcription (RT-PCR) of
the mRNA of interest provides a means for measuring gene expression
using as few as one cell. When RT-PCR is performed, the reliability of
the data can be highly subjective due to the efficiency of both
RT and PCR steps.
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12. Real-time PCR steps
The first step in a real-time PCR reaction is the conversion of RNA to
complementary DNA (cDNA) – this process is known as reverse
transcription.
The next step uses fluorescent reporters and a PCR reaction to
amplify and detect specific genes.
Two types of fluorescent reporters are commonly used;
SYBR green & TaqMan probes.
SYBR green is a dye that fluoresces only when bound to double
stranded DNA (i.e., the PCR product).
TaqMan probes are made of a gene-specific nucleic acid probe, joined
to reporter and quencher molecules.
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14. Applications of PCR
Medicine:
• Testing of genetic disease mutations.
• Monitoring the gene in gene therapy.
• Detecting disease-causing genes in the parents.
Forensic Science:
• Used as a tool in genetic fingerprinting.
• Identifying the criminal from millions of people.
Research and Genetics:
• Compare the genome of two organisms in genomic studies.
• In the phylogenetic analysis of DNA from any source such as fossils.
• Analysis of gene expression.
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