PCR introduction, history, enzymes in PCR, Reaction components, PCR cycle, steps involved in PCR, RT-PCR, Applications.

1. POLYMERASE CHAIN
REACTION
Presented by~
B. Likitha
M. Pharmacy I-I
Pharmaceutical Analysis
Advanced Pharmaceutical Analysis

2. INTRODUCTION
• PCR, polymerase chain reaction, is an in-vitro technique for
amplification of a region of DNA whose sequence is known or
which lies between two regions of known sequence.
• Before PCR, DNA of interest could only be amplified by over-
expression in cells and this with limited yield.

3. • 1966, Thomas Brock discovered Thermus Aquaticus, a
thermostable bacteria in the hot springs of Yellowstone
National Park.
• 1983, Kary Mullis postulated the concept of PCR ( Nobel
Prize in 1993).
• 1985, Saiki publishes the first application of PCR ( beta-
Globin).
• 1985, Cetus Corp. Scientists isolate Thermostable Taq
Polymerase (from T.Aquaticus), which revolutionized
PCR.
HISTORY

4. DNA REPLICATION VS. PCR
• PCR is a laboratory version of DNA Replication in cells.
• The laboratory version is commonly called “in vitro” since
it occurs in a test tube while “in vivo” signifies occurring in
a living cell.

5. DNA REPLICATION IN CELLS
(IN VIVO)
• DNA replication is the copying of
DNA.
• It typically takes a cell just a few
hours to copy all of its DNA.
• DNA replication is semi-
conservative (i.e. one strand of the
DNA is used as the template for
the growth of a new DNA strand).
• This process occurs with very few
errors (on average there is one
error per 1 billion nucleotides
copied).
• More than a dozen enzymes and
proteins participate in DNA
replication.

6. Key enzymes involved in DNA
Replication
• DNA Polymerase
• DNA Ligase
• Primase
• Helicase
• Topoisomerase
• Single strand binding protein

7. DNA REPLICATION ENZYMES
1. DNA POLYMERASE
• Catalyzes the elongation of DNA by adding nucleoside
triphosphates to the 3’ end of the growing strand.
• A nucleotide triphosphate is a 1 sugar + 1 base + 3
phosphates.
• When a nucleoside triphosphate joins the DNA strand, two
phosphates are removed.
• DNA polymerase can only add nucleotides to 3’ end of
growing strand.

8. Complementary Base-Pairing in DNA
• DNA is a double helix, made up of nucleotides, with a
sugar-phosphate backbone on the outside of the helix.
• Note: a nucleotide is a sugar + phosphate + nitrogenous
base
• The two strands of DNA are held together by pairs of
nitrogenous bases that are attached to each other via
hydrogen bonds.
• The nitrogenous base adenine will only pair with thymine
• The nitrogenous base guanine will only pair with cytosine
• During replication, once the DNA strands are separated,
DNA polymerase uses each strand as a template to
synthesize new strands of DNA with the precise,
complementary order of nucleotides.

9. DNA Replication enzymes
2. DNA LIGASE
• The two strands of DNA in a double helix are antiparallel
(i.e. they are oriented in opposite directions with one
strand oriented from 5’ to 3’ and the other strand oriented
from 3’ to 5’
• 5’ and 3’ refer to the numbers assigned to the carbons in
the 5 carbon sugar
• Given the antiparallel nature of DNA and the fact that
DNA ploymerases can only add nucleotides to the 3’ end,
one strand (referred to as the leading strand) of DNA is
synthesized continuously and the other strand (referred to
as the lagging strand) is synthesized in fragments (called
Okazaki fragments) that are joined together by DNA
ligase.

10. DNA Replication enzymes
3. PRIMASE
• DNA Polymerase cannot initiate the synthesis of DNA
• Remember that DNA polymerase can only add nucleotides
to 3’ end of an already existing strand of DNA
• In humans, primase is the enzyme that can start an RNA
chain from scratch and it creates a primer (a short stretch
RNA with an available 3’ end) that DNA polymerase can
add nucleotides to during replication.
Note that the RNA primer is subsequently
replaced with DNA

11. DNA Replication enzymes
(Helicase, Topoisomerase and Single-strand binding
protein)
• Helicase untwists the two parallel DNA strands
• Topoisomerase relieves the stress of this twisting
• Single-strand binding protein binds to and stabilizes the
unpaired DNA strands

12. PCR: the in vitro version of DNA
Replication
The following components are needed to perform PCR in the
laboratory:
1) DNA (your DNA of interest that contains the target sequence
you wish to copy)
2) A heat-stable DNA Polymerase (like Taq Polymerase)
3) All four nucleotide triphosphates
4) Buffers
5) Two short, single-stranded DNA molecules that serve as
primers
6) Thin walled tubes
7) Thermal cycler (a device that can change temperatures
dramatically in a very short period of time)

13. Reaction Components
• DNA template
• Primers
• Enzyme
• dNTPs
• Mg2+
• buffers

14. What is PCR? :
The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates (DNA building blocks).
4) Thermostable DNA Polymerase - enzyme that
catalyzes the reaction.
5) Mg++ ions - cofactor of the enzyme.
6) Buffer solution – maintains pH and ionic strength of the
reaction solution suitable for the activity of the enzyme.

15. 1. DNA template
• DNA containing region
to be sequenced
• Size of target DNA to
be amplified : up to 3
Kb

16. 2. Primers
• 2 sets of primers
• Generally 20-30
nucleotides long
• Synthetically produced
• complimentary to the 3’
ends of target DNA
• not complimentary to each
other

17. Primers (ctnd)
• Not containing inverted repeat sequences to avoid
formation of internal structures.
• 40-60% GC content preferred for better annealing.
• Tm of primers can be calculated to determine annealing T0.
• Tm = 0.41(%G+C) + 16.6 log(J+) + 81.5 where J+ is the
concentration of monovalent ions.

18. 3. Enzyme
• Usually Taq Polymerase or anyone of the natural or
Recombinant thermostable polymerases
• Stable at T0 up to 950 C
• High processivity
• Taq Pol has 5’-3’ exo only, no proof reading

19. Heat-stable DNA Polymerase
• Given that PCR involves very high temperatures, it is
imperative that a heat-stable DNA polymerase be used in
the reaction.
• Most DNA polymerases would denature (and thus not
function properly) at the high temperatures of PCR.
• Taq DNA polymerase was purified from the hot springs
bacterium Thermus aquaticus in 1976
• Taq has maximal enzymatic activity at 75 C to 80 C, and
substantially reduced activities at lower temperatures.

20. The PCR Cycle
• Comprised of 3 steps:
1. Denaturation of DNA at
950C
2. Primer hybridization
(annealing) at 40-500C
3. DNA synthesis ( Primer
extension) at 720C
PCR Thermocycler

21. Thermal cycler
 Earliest thermal cyclers were designed for use with the Klenow
fragment of DNA polymerase I.
 Peltier element - Modern PCR machines.
 Lid temperature - 105oC.
 Thermal blocks - 48/96 wells.
The thermal cycler (also known as a thermocycler, PCR
machine or DNA amplifier) is a laboratory apparatus most commonly
used to amplify segments of DNA via the polymerase chain
reaction (PCR). Thermal cyclers may also be used in laboratories to
facilitate other temperature-sensitive reactions, including restriction
enzyme digestion or rapid diagnostics. The device has a thermal
block with holes where tubes holding the reaction mixtures can be
inserted. The cycler then raises and lowers the temperature of the block
in discrete, pre-programmed steps.

22. Step 1: Denaturation. As in DNA replication, the
two strands in the DNA double helix need to be
separated.

23. Step 2: Annealing. Primers bind to the
target DNA sequences and initiate
polymerization.

24. Step 3: Extension. New strands of DNA
are made using the original strand as
templates.

27. RT-PCR
• Reverse Transcriptase PCR
• Uses RNA as the initial template
• RNA-directed DNA polymerase (rTh)
• Yields ds cDNA

31. Detection of amplification
products
• Gel electrophoresis
• Sequencing of amplified fragment
• Southern blot

32. Applications
• Genome mapping and gene function determination
• Biodiversity studies ( e.g. evolution studies)
• Diagnostics ( prenatal testing of genetic diseases, early
detection of cancer, viral infections...)
• Detection of drug resistance genes
• Forensic (DNA fingerprinting)

33. PCR and Disease
• Primers can be created that will only bind and amplify
certain alleles of genes or mutations of genes
• This is the basis of genetic counseling and PCR is used as
part of the diagnostic tests for genetic diseases.
• Some diseases that can be diagnosed with the help of PCR:
– Huntington's disease
– Cystic fibrosis
– Human immunodeficiency virus (HIV)

34. Huntington’s Disease (HD)
• HD is a genetic disorder characterized by abnormal body
movements and reduced mental abilities
• HD is caused by a mutation in the Huntingtin (HD) gene
• In individuals with HD, the HD gene is “expanded”
– In non-HD individuals, the HD gene has a pattern called
trinucleotide repeats with “CAG” occurring in repetition
less than 30 times.
– IN HD individuals, the “CAG” trinucleotide repeat occurs
more that 36 times in the HD gene
• PCR can be performed on an individual’s DNA to determine
whether the individual has HD.
– The DNA is amplified via PCR and sequenced (a technique
by which the exact nucleotide sequence is determined) and
the number of trinucleotide repeats is then counted.

35. Cystic Fibrosis (CF)
• CF is a genetic disease characterized by severe breathing
difficulties and a predisposition to infections.
• CF is caused by mutations in the cystic fibrosis
transmembrane conductance regulator (CTFR) gene.
• In non-CF individuals, the CTFR gene codes for a protein
that is a chloride ion channel and is involved in the
production of sweat, digestive juices and mucus.
• In CF individuals, mutations in the CTFR gene lead to
thick mucous secretions in the lungs and subsequent
persistent bacterial infections.
• The presence of CTFR mutations in a individual can be
detected by performing PCR and sequencing on that
individual’s DNA.

36. Human Immunodeficiency Virus
(HIV)
• HIV is a retrovirus that attacks the immune system.
• HIV tests rely on PCR with primers that will only amplify
a section of the viral DNA found in an infected
individual’s bodily fluids.
Therefore if there is a PCR product, the person is likely to
be HIV positive. If there is no PCR product the person is
likely to be HIV negative.
• Protein detection based tests are available as well but all
US blood is tested by PCR.

37. PCR Applications to Forensic
Science
• Paternity suits -Argentina’s Mothers of the plaza and their
search for abducted grandchildren.
• Identifying badly decomposed bodies or when only body
fragments are found - World trade center, Bosnian , Iraq &
Rwandan mass graves.

38. Advantages
• Automated, fast, reliable (reproducible) results
• Contamination (less chances of contamination)
• High output
• Sensitive
• Broad uses
• Defined, easy to follow protocols

39. Conclusion
The speed and ease of use, sensitivity, specificity and
robustness of PCR has revolutionised molecular biology
and made PCR the most widely used and powerful
technique with great spectrum of research and diagnostic
applications.

40. Thank you