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 68ºC, whereas the method followed here used an annealing temperature of 60ºC.

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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 95ºC in order to denature the DNA – that is to

separate the two strands.8. The temperature is then lowered to 60ºC.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 72ºC 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 ¼.12. After 25 cycles the reaction tube contains 33,554,432 of the target DNA.

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