this doc is having basic information about PCR techmique. it contains history, principle, advantages, disadvantages, and applications. it can give a brief idea about pcr technique.

1. Jaimini v.
patoliya

3.  PCR is a technique that takes specific
sequence of DNA of small amount and
amplifies it to be used for further testing.
 In vitro technique.
 A photocopier.

4.  Method first proposed by H. G. Khorana & colleagues
in 1970’s.
 15 years later the idea was independently conceived by
 Karry Mullis in 1983.
Used the Klenow fragment of E. coli DNA
polymerase to describe the in-vitro amplification of
genes.
 awarded the Noble prize for chemistry in 1993.
 Saiki et al in 1988
used the thermostable DNA polymerase from
Thermus aquaticus and greatly increased the efficiency
 1988: PerkinElmer introduces the automated
thermal cycler.

5.  In 1989, Science magazine selected PCR as the major
scientific development and Taq DNA polymerase as
the molecule of the year.
 1990: amplification and detection of specific DNA
sequences using a fluorescent DNA-binding dye,
laying the foundation for future "real-time" or
"kinetic" PCR.
 1991: RT-PCR is developed using a single
thermostable polymerase, rTth, facilitating
diagnostic tests for RNA viruses.

7.  The technique has been a revolution in molecular
biology and now it is difficult to imagine life without
it.
 The problem of insufficient DNA is no longer a
problem in molecular biology research or DNA based
diagnostics.
 Most PCR methods amplify DNA fragments of
between 0.1 and 10 kilo base pairs.

9.  a DNA template
 a DNA polymerase - heat-resistant Taq
polymerase
 two DNA primers that are complementary to
the 3‘ ends of each of the sense and anti-
sense strands of the DNA
 deoxynucleoside triphosphates, or dNTPs -the
building blocks
 a buffer solution
 bivalent cations, typically magnesium (Mg)
or manganese (Mn) ions; Mg2+ is the most
common.

10.  The reaction is commonly carried out in a
volume of 10–200 μl in small reaction tubes
(0.2–0.5 ml volumes) in a thermal cycler.

13.  Initialization:
• for DNA polymerases that require heat activation.
• 94–96 °C for 1–10 minutes.
 Denaturation:
• first regular cycling event
• 94–98 °C (201–208 °F) for 20–30 seconds.
• DNA melting, or denaturation.
 Annealing:
• 50–65 °C (122–149 °F) for 20–40 seconds,
• allowing annealing of the primers to each single-
stranded DNA templates.
• A typical annealing temperature is about 3–5 °C below
the Tm of the primers used.

14.  Extension/elongation:
• The temperature at this step depends on the DNA
polymerase used.
• 75–80 °C.
• condensing the 5'-phosphate group of the dNTPs with
the 3'-hydroxy group at the end of the nascent
(elongating) DNA strand. The processes of
denaturation, annealing and elongation constitute a
single cycle.
• The formula used to calculate the number of DNA
copies formed after a given number of cycles is 2n,
where n is the number of cycles. Thus, a reaction set
for 30 cycles results in 230.

15.  Final elongation:
• 70–74 C° for 5–15 minutes after the last PCR cycle to
ensure that any remaining single-stranded DNA is fully
elongated.
 Final hold:
• The final step cools the reaction chamber to 4–15 °C
for an indefinite time
• may be employed for short-term storage of the PCR
products.

18.  Small amount of DNA is required.
 Result obtained more quickly - within 1 day.
 not necessary to use radioactive material.
 PCR is much more precise in determining the
sizes of alleles - essential for some disorders.
 Large numbers of copies of specific DNA
sequences can be amplified simultaneously with
multiplex PCR reactions.
 Contaminant DNA, such as fungal and bacterial
sources, will not amplify because human-specific
primers are used.

19.  One major limitation of PCR is that prior
information about the target sequence is
necessary in order to generate the primers
that will allow its selective amplification.
 False positive results (cross contamination).
 False negative results

20.  IN RESEARCH:
 Generation of hybridization probes for Southern or northern
hybridization and DNA cloning, cDNA libraries.
 in research laboratories in DNA sequencing, recombinant
DNA technology.
 Major role in the human genome project.
 Medical applications
 Infectious disease applications
 identification of non-cultivatable or slow-growing
microorganisms such as mycobacteria, anaerobic bacteria,
or viruses i.e. human immunodeficiency
virus , tuberculosis.

21.  Single point mutations can be detected by modified PCR
techniques such as the ligase chain reaction (LCR) and
PCR- single-strand conformational polymorphisms
(PCR-SSCP)
 Detection of variation and mutation in genes using primers
 Cancer -early diagnosis of malignant diseases such
as leukemia and lymphoma.
 Prenatal sexing :Test for x-linked recessive genetic disorders
i.e. sickle cell anemia, thalassamia.
 essential to preimplantation genetic diagnosis.
 Prenatal Diagnosis of diseases: Prenatal diagnosis of many of
the inborn errors of metabolism is possible by DNA markers.

22.  Forensic applications
 when only a trace amount of DNA is available as
evidence.
 PCR may also be used in the analysis of ancient
DNA that is tens of thousands of years old.
 genetic fingerprinting from Minute samples of DNA
can be isolated from a crime scene, and compared to
that from suspect.
 DNA paternity testing.