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Mls 522 Assignment

Talks about molecular biology

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Mayowa Ogunmola
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9 views

Mls 522 Assignment

Talks about molecular biology

Uploaded by

Mayowa Ogunmola
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Name: Olaitan kehinde Sarah

Matric number: 2008010006

Course: Mls 522.

Specialty: medical microbiology.

Describe PCR and it's application

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.

Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA
sequences. It allows researchers to create millions to billions of copies of a particular DNA segment from
a small initial sample. The process involves repeated cycles of heating and cooling, which facilitate the
DNA denaturation, annealing of primers to the target sequence, and extension of the new DNA strand
by a DNA polymerase enzyme.

Steps of PCR:

Denaturation: The double-stranded DNA is heated to around 94-98°C to separate it into two single
strands.

Annealing: The temperature is lowered to around 50-65°C to allow primers to bind to their
complementary sequences on the single-stranded DNA.
Extension: The temperature is raised to about 72°C, the optimal temperature for the DNA polymerase to
synthesize a new DNA strand by adding nucleotides to the primers.

Step 1: Denaturing

The reaction mixture is heated to 94-95⁰C, for between 15 and 30 seconds.

The high temperature causes the hydrogen bonds between the bases in two strands of template DNA to
break and the two strands to separate.

This results in two single strands of DNA, which will act as templates to produce the new copies of each
strand of DNA.

It is important that the temperature is maintained at this stage for long enough to ensure that the DNA
strands have separated completely.

Step 2: Annealing

The reaction is cooled to enable the primers to attach to a specific location on the single-stranded
template DNA by way of hydrogen bonding.

The temperature depends on the characteristics of the primer, but is usually between 50 and 65⁰C.

The two separated strands of DNA are complementary and run in opposite directions (from one end –
the 5’ end – to the other – the 3’ end). As a result, there are two primers – a forward primer and a
reverse primer.

This step is essential because the primers serve as the starting point for DNA synthesis, by providing a
short region of double stranded DNA for the polymerase enzyme to work with. Only once the primer has
bound can the polymerase enzyme attach and start making the new complementary strand of DNA from
the loose DNA bases, in the extending step.

The annealing step usually takes about 10-30 seconds.

Step 3: Extending

The heat is increased to 72⁰C to enable the new DNA to be made by a special Taq DNA polymerase
enzyme which adds DNA bases.

Taq DNA polymerase is an enzyme taken from the bacteria Thermus aquaticus (“Taq”):
This bacterium normally lives in hot springs so can tolerate temperatures above 80⁰C, but its optimum
temperature is 72⁰C.

The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the
temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.

DNA polymerase from most other organisms would not be able to withstand these high temperatures.
For example, human polymerase works ideally at 37˚C (body temperature).

At 72⁰C, the Taq polymerase begins to build the complementary strand. It attaches to the primer and
then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction.

The result is a brand-new strand of DNA and a double-stranded molecule of DNA.

The duration of this step depends on the length of DNA sequence being amplified. It usually takes
around one minute to copy 1,000 DNA bases.

Applications of PCR:

Medical Diagnostics: Used to detect the presence of pathogenic DNA or RNA in a patient's sample, such
as in the diagnosis of infectious diseases (e.g., COVID-19, HIV).

Genetic Testing: Identifies genetic mutations associated with hereditary diseases.

Forensic Science: Amplifies DNA from crime scene evidence for identification purposes.

Research: Studies gene expression, genetic mutations, and genetic mapping.

Cloning: Amplifies DNA fragments for cloning into vectors.

Evolutionary Biology: Analyzes genetic material from ancient or degraded samples to study evolutionary
relationships.

PCR has revolutionized molecular biology and genetics by providing a fast, efficient, and reliable method
to amplify DNA, enabling a wide range of scientific and medical advancements.

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