Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment. This slides introduces pcr importances ,uses and steps of pcr.

1. POLYMERASE CHAIN REACTION(PCR)
PRESENTED BY
SUBINA SUNAR

2. Polymerase Chain Reaction (PCR)
◦ Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing
(amplifying) millions to billions of copies of a specific segment of DNA, which can then be
studied in greater detail. PCR involves using short synthetic DNA fragments called primers to
select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to
amplify that segment.

3. ◦ Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA
region in vitro (in a test tube rather than an organism).
◦ In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow
many copies of the target region to be produced.
◦ PCR has many research and practical applications. It is routinely used in DNA cloning, medical
diagnostics, and forensic analysis of DNA.

4. ◦ Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies
(millions or billions!) of a particular region of DNA. This DNA region can be anything the
experimenter is interested in. For example, it might be a gene whose function a researcher wants
to understand, or a genetic marker used by forensic scientists to match crime scene DNA with
suspects.

5. ◦ Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or
used in some other way. For instance, DNA amplified by PCR may be sent for sequencing,
visualized by gel electrophoresis, or cloned into a plasmid for further experiments.
◦ PCR is used in many areas of biology and medicine, including molecular biology research,
medical diagnostics, and even some branches of ecology.

6. HISTORY
◦ Kary Mullis invented the PCR technique in 1985
while working as a chemist at the
Cetus Corporation, a biotechnology firm in
Emeryville, California. The procedure requires
placing a small amount of the DNA containing
the desired gene into a test tube.
◦ For this work , Mullis received 1993 Noble Price
in chemisty.

7. TYPES OF PCR
◦ Real-Time PCR (quantitative PCR or qPCR) – in which DNA molecules are tagged using fluorescent dye,
which is used to monitor and quantify PCR products in real-time
◦ Reverse-Transcriptase (RT–PCR) – creates complementary DNA (cDNA) by reverse transcribing RNA to
DNA using reverse transcriptase
◦ Multiplex PCR – uses a number of primers to multiply multiple fragments in a single DNA sample
◦ Nested PCR – after the initial 25-35 PCR cycles, an additional PCR is conducted using new primers
“nested” within the original primers, which reduces the risk of unwanted products
◦ Hot Start PCR – in which heat is used to denaturate antibodies that are used to inactivate Taq polymerase
◦ Long-range PCR – longer ranges of DNA are formed by using a mixture of polymerases
◦ Assembly PCR – longer DNA fragments are aplified by using overlapping primers
◦ Asymmetric PCR – only one strand of the target DNA is amplified
◦ In situ PCR – PCR that takes place in cells, or in fixed tissue on a slide

8. PCR USED FOR
 Genotyping
 Cloning
 Mutation detection
 Sequencing
 Microarrays
 Forensics
 Paternity testing

9. STEPS OF PCR
The basic steps are given below :
1. Denaturation (96 ℃ ): Heat the reaction strongly to separate, or
denature, the DNA strands. This provides single-stranded template for the next steps.
2. Annealing (55-65 ℃ ): Cool the reaction so the primers can bind to their complementary sequence on the
single-stranded template DNA.
3. Extension (72 ℃ ): Raise the reaction temperature so Taq polymerase extends the primers, synthesizing
new strands of DNA

10. o This cycle repeats
25 - 35 times in a typical
PCR reaction, which
generally takes 2 -
4 hours, depending on
the length of the DNA
region being copied.
If the reaction is efficient
(works well), the target
region
can go from just one or a
few copies to billions.
.

11. PCR CYCLE

12. PCR STEPS Vs TEMPERATURE

13. PCR COMPONENTS REACTION
1. Water
2. Buffer
3. DNA template
4. Primers
5. Nucleotides
6. Mg++ ions
7. DNA polymerase

14. 1.Water
◦ The medium for all other components.
2.Buffer
◦ Stabilizes the DNA polymerase, DNA, and nucleotides
◦ 500 mM KCl
◦ 100 mM Tris-HCl, pH 8.3
◦ Triton X-100 or Tween

15. 3.DNA template
◦ Contains region to be amplified
◦ Any DNA desired
◦ Purity not required
◦ Should be free of polymerase inhibitors
4.Primers
◦ Specific for ends of amplified region
◦ Forward and Reverse
◦ Annealing temps should be known
◦ Depends on primer length, GC content, etc.
◦ Length 15-30 nt
◦ Conc 0.1 – 1.0 uM (pMol/ul)

16. 5.Nucleotides
◦ Added to the growing chain
◦ Activated NTP’s
◦ dATP, dGTP, dCTP, dTTP
◦ Stored at 10mM, pH 7.0
◦ Add to 20-200 uM in assay
6.Mg++ ions
◦ Essential co-factor of DNA polymerase
◦ Too little: Enzyme won’t work.
◦ Stabilizes the DNA double-helix
◦ Too much: DNA extra stable, non-specific priming, band smearing
◦ Used at 0.5 to 3.5 uM in the assay

17. 7.DNA Polymerase
◦ The enzyme that does the extension
◦ TAQ or similar
◦ Heat-stable
◦ Approx 1 U / rxn

18. FACTORS AFFECTING THE ACCURACY AND
TIME FOR PCR
• Annealing temperature: As discussed above, the annealing temperature
needs to be maintained. If the temperature is too low then there can be
mismatch pairing. The mismatched pairing can lead to decrease in the
quality of the PCR product.
• Length of the primer used: The primer used needs to be unique. If the
primer used has a very short sequence then there are chances that it
would bind randomly to any non-target sequence. If the sequence of the
primer is long then its specificity to bind target sequence increase.
However, if the primer is too long, then it would take a long time to find
and anneal to the target sequence.

19. ADVANTAGES OF PCR
1. Highly specific: PCR can distinguish DNA sequences by just one nucleotide, making it a very
accurate technique.
2. Sensitive: PCR is a very useful technique when the amount of DNA sample is limited because it
allows the detection of even a single copy of a specific DNA template.
3. Versatile: The PCR technique can be used for various applications like genetic testing, criminal
investigations, and paternity tests.
4. Rapid and efficient: PCR can efficiently and rapidly amplify a small amount of DNA sample to
million copies in just a few hours.

20. DISADVANTAGES OF PCR
1. Contamination: The PCR technique is very susceptible to contamination from other sources of DNA or
RNA or the environment. This can mislead data interpretation.
2. Cost and complexity: PCR can be expensive and requires expert knowledge for high-throughput projects.
3. Lack of novel information: Since PCR can only amplify and target specific DNA sequences targeted by the
primers, PCR provides limited information and cannot detect novel DNA sequences.
4. Inhibition from sample content: The whole PCR cycle can be disrupted by inhibitors that co-purify with
DNA, such as heme from blood samples, reducing the sensitivity of the process.
5. Errors in amplification: Base substitutions, indels, and other alterations in DNA sequences can lead to
inaccurate amplification and hence, false results.

21. HOW PCR TEST IS DONE
◦ The COVID-19 test is done by taking a swab of the back of your throat and your nose.
◦ COVID-19 testing uses a modified version of PCR called quantitative polymerase chain reaction
(qPCR). This method adds fluorescent dyes to the PCR process to measure the amount of
genetic material in a sample. In this instance, healthcare workers measure the amount of genetic
material from SARS-CoV-2.

22. PCR INSTRUMENT USED

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