Benedicte Symcox

Student number: SS127293

Core Practical 6.1

8th July 2014

DNA Amplification using PCR

Introduction
DNA profiling is used to identify an individual from small fragments of DNA. The
DNA is initially extracted from a small tissue sample, and must then be copied, or
amplified in order to provide enough material for the identification processes.
When DNA replicates, its two strands separate, and complementary nucleotides
line up by the original strands. Hydrogen bonds then form between the
complementary bases and the nucleotides join together to form a new double
strand of DNA. The enzyme DNA polymerase allows this process to occur.
DNA polymerase requires a primer to know where to begin the replication
process. This primer is a short piece of single stranded DNA complementary to
the start of the target DNA. If tagged with a fluorescent marker, the primers also
allow for the detection of the amplified DNA target fragments which is essential
in the next steps of DNA profiling.
Method
DNA is extracted from biological material and the resulting sample can be cut
into fragments using restriction endonuclease enzymes.
Many copies of these fragments can be made using the PCR reaction, which
involves a repeated cycle of temperature changes. Each cycle doubles the
number of copies produced. The exact temperatures vary according to the
species of DNA being amplified. This diagram shows an annealing temperature of
68C, whereas the method followed here used an annealing temperature of 60C.

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Benedicte Symcox
Student number: SS127293

Core Practical 6.1

8th July 2014

For the reaction to be successful, four elements are required: the target DNA
sample, free nucleotides, taq polymerase and primers.

Understanding your method

1. A buffer solution is used to maintain pH during DNA extraction.
2. The detergent disrupts cell membranes, allowing the DNA to be released
into the buffer solution.
3. Other than the target DAN, the other ingredients of the PCR mixture are
free nucleotides, primers and Taq polymerase.
4. A DNA primer is typically 20 nucleotides long.
5. The enzyme Taq polymerase is particularly suitable for the PCR reaction
because it tolerates the high temperatures involved in the PCR reaction.
6. The PCR reaction lasts for several hours as the cycle is repeated between
25 and 30 times..
7. At the beginning, the temperature is raised to 95C in order to denature
the DNA that is to separate the two strands.
8. The temperature is then lowered to 60C.
9. The primers form hydrogen bonds with the target DNA at this temperature
in a process known as annealing.
10.The temperature is then raised again to 72C because this is the optimal
temperature at which Taq polymerase functions.
11.After three cycles, only 2 out of the 8 copies contain only the target DNA
with no flanking regions a proportion of .
12.After 25 cycles the reaction tube contains 33,554,432 of the target DNA.

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