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Polymerase chain reaction

- This is a technique that makes many copies of DNA in order to effectively run the DNAprofiling.

- PCR provides an in-vitro means of making many copies of DNA unlike the in-vivo (cloning).- PCR has advantages over cloning in bacteria as follows;

(i) Bacterial cloning is slowertakes several days yet PCR takes few hours(ii)

More errors in bacterial cloning due to mutations.

(iii) Bacterial cloning is difficult

In PCR, primers are used. The structure of the primer is as follows;

- Single stranded short sections of DNA that consist of 10-40 nucleotides.- These nucleotides are complementary to those on target length of DNA strand.- The function of primers is;

It binds to target length of DNA strand to start polymerase reaction.

The PCR cycle involves the following stages;

(i) The DNA sample is heated to 90-950c for 20 seconds. This breaks the hydrogen bondsbetween the bases (nucleotides) to separate the strands in order to expose the base

sequences.

(ii) The sample is cooled to between 550c to 600c for 20 seconds. The primers and nucleotidesare added. This temperature allows the primers to anneal (join) to the complementary

strands of DNA by hydrogen bonds which are formed between the base pairs. Also

nucleotides bind with the complementary nucleotides of the DNA strand by hydrogen

bonds starting from the end of the primer.

(iii) The sample is heated to 750c for 30 seconds. It allows the DNA polymerase to bind atthe end of primers so that it catalyses the formation of phophodiester bonds between the

nucleotides. This extends the primer and restores the double strand of DNA.

(iv) Stages i iii are repeated about 30 times to produce about 1 billion copies of theoriginal DNA. The whole process takes about 3 hours.

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DNA Digestion

- This is where restriction enzymes cut the DNA molecules.- Double stranded DNA molecules are mixed with restriction enzymes and DNA is cut into

different sizes of fragments.

- Restriction enzymes cut either side of STRs.

Gel electrophoresis

- This is a technique that separates charged molecules in an electric field based on their charge andsize.

- The positively charged molecules move to the cathode.- The negatively charged molecules move to the anode.- The smallest molecules move far away from the wells towards electrodes and vice versa.- The DNA sample is mixed with a dye that does not bind with the DNA. The dye moves through

the gel faster than the DNA so that the current can be switched off to stop the samples from

running off the end.

- The DNA sample mixed with this dye is placed in wells in a buffer solution on a layer of gel.- Electrodes are arranged so that a direct current is passed through this medium.- When the current is switched on, the substances are separated based on their charge and size.- For the DNA molecule, being negatively charged, the fragments move towards positively charged

electrode called anode.

- The fragments separate into invisible bands.