- Created by: H M
- Created on: 10-03-19 14:38
- Analyses the pattern of Short Tandem Repeats in a limited number of introns
1. Extract the DNA sample (from fossil/ from blood, semen, saliva, skin cells in fur or rectal cells in feces)
2. Amplify DNA using PCR (Polymerase Chain Reaction).
PCR mixture contains
~ Sample of DNA to be amplified
~ Excess of appropriate primers (short single-stranded sequences of DNA made of 10-40 deoxyribonucleotides. They have a base sequence complementary to one end of the target DNA strand and bind to the start of the STR sequence. Required to initiate the PCR as DNA polymerase cannot start replication from scratch.
~ DNA polymerase enzyme (should be heat stable and needs a primer to initiate DNA synthesis)
~Good supply of the 4 types of deoxyribonucleotides
~ Buffer of pH7
PCR mixture is
i. Heated to 93 oC for 1min
~ Break hydrogen bonds between complementary bases and separate complementary strands of DNA
~ Both strands act as templates for the formation of complementary DNA strands
ii. Cooled to 55 oC for 2mins
~ Primers join to the start of the STR sequence
iii. Heated to 75 oC for 2mins
~ DNA polymerase attaches to the primer
~ A complementary copy of each DNA strand is built by extending the primers
· Individual mononucleotides line up against the template according to the complementary base pairing rule. Adjacent mononucleotides are joined by phosphodiester bonds, which is catalyzed by DNA polymerase.
iv. Cycle is repeated 25-30 times to produce 225-30copies…