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PCR Group Assignment

The document discusses the history and evolution of the PCR technique, developed in the 1980s by Kary Mullis and others, which revolutionized various fields such as forensic science and diagnostic medicine. It outlines the principles and procedures of PCR, including denaturation, annealing, and extension, leading to exponential amplification of DNA. Additionally, it briefly mentions related techniques like Western, Southern, and Northern blotting, highlighting their applications in detecting proteins and RNA.

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8 views15 pages

PCR Group Assignment

The document discusses the history and evolution of the PCR technique, developed in the 1980s by Kary Mullis and others, which revolutionized various fields such as forensic science and diagnostic medicine. It outlines the principles and procedures of PCR, including denaturation, annealing, and extension, leading to exponential amplification of DNA. Additionally, it briefly mentions related techniques like Western, Southern, and Northern blotting, highlighting their applications in detecting proteins and RNA.

Uploaded by

belinda.nyagah
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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PCR GROUP

ASSIGNMENT.
JOSEPH MUTIKA
PHILLIP WESONGAH
BRIAN LANGAT
MONICA MORAA
BELINDA NYAGA
PCR HISTORY AND EVOLUTION.
• The PCR technique as we know today was
conceptualized and developed in the 1980s by Kary
Mullis and his colleagues at Cetus Corporation. The
isolation and purification of thermostable Taq
polymerases led to the automation of the initially slow
and laborious PCR technique and the development of
programmable PCR thermal cyclers made it a widely
used technique at many levels of biology and
chemistry.
• Although Mullis invented the PCR, its successful
applications in several fields are a result of a lot of
hard work by other Cetus researchers such as Henry
Erlich. Also, there are challenges to the PCR patents
held by Mullis based on early research works
performed by Khorana et al. in the 1960s and 1970s.
….
• Thanks to its extraordinary versatility, today, PCR is
being used in diverse fields such as forensic science,
environmental studies, food technology, and
diagnostic medicine. The massive advancement over
the years in our understanding of the genome of
humans and several other species would not have
been possible without the remarkable yet simple
technique called PCR.
PCR PRINCIPLE.
• 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.
PCR PROCEDURES AND STEPS
1.DENATURATION
The DNA template is heated to 94° C. This breaks the
weak hydrogen bonds that hold DNA strands together
in a helix, allowing the strands to separate creating
single stranded DNA.
2.ANEALING.
• The mixture is cooled to anywhere from 50-70°
C. This allows the primers to bind (anneal) to
their complementary sequence in the template
DNA.
3.EXTENSION.
• The reaction is then heated to 72° C, the optimal temperature for
DNA polymerase to act. DNA polymerase extends the primers, adding
nucleotides onto the primer in a sequential manner, using the target
DNA as a template.

• With one cycle, a single segment of double-stranded DNA template is


amplified into two separate pieces of double-stranded DNA. These
two pieces are then available for amplification in the next cycle. As
the cycles are repeated, more and more copies are generated and the
number of copies of the template is increased exponentially.
APPLICATIONS OF PCR
• PCR is used in analyzing clinical specimens for the
presence of infectious agents, including HIV, hepatitis,
malaria, anthrax, etc.
• PCR can provide information on a patient’s prognosis,
and predict response or resistance to therapy. Many
cancers are characterized by small mutations in
certain genes, and this is what PCR is employed to
identify.
• PCR is used in the analysis of mutations that occur in
many genetic diseases (e.g. cystic fibrosis, sickle cell
anaemia, phenylketonuria, muscular dystrophy).
• PCR is also used in forensics laboratories and is
especially useful because only a tiny amount of
original DNA is required, for example, sufficient DNA
can be obtained from a droplet of blood or a single
hair.
• PCR is an essential technique in cloning procedure
which allows generation of large amounts of pure
DNA from tiny amount of template strand and further
study of a particular gene.
• The Human Genome Project (HGP) for determining
the sequence of the 3 billion base pairs in the human
genome, relied heavily on PCR.
• PCR has been used to identify and to explore
relationships among species in the field of
evolutionary biology. In anthropology, it is also used to
understand the ancient human migration patterns. In
archaeology, it has been used to spot the ancient
human race. PCR commonly used by Paleontologists
to amplify DNA from extinct species or cryopreserved
fossils of millions years and thus can be further
studied to elucidate on.
WESTERN,SOUTHERN AND
NORTHERN BLOTTING.
• Western blotting is the counterpart which is
used to detect proteins. The difference lies in the
visualization process. In Western blotting, this is
made possible by primary and secondary
antibodies, whereas in Southern blotting, a
radiolabeled (fluorescent) probe or dye that
binds to the DNA is used.
• Application of Western blotting includes identifying
HIV antigens or Hepatitis B surface antigen in blood.
Northern blotting is used to detect mRNA of interest,
where after separation by electrophoresis, cDNA is
used as a probe that binds to the RNA strand; the
application includes finding alternate transcript size.
The term ‘blotting’ in all the three techniques
represents the transfer of material after separation to
nitrocellulose paper by means of diffusion.
•THANK YOU

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