Method for Core Practical
Preparing a DNA profile
Match the descriptions of each sequence with the correct stage on the diagram flowchart. You could cut out the flow chart and stick the correct description next to each picture to provide a summary of DNA profiling.
A. Denaturation: the double-stranded DNA fragments are immersed in alkali to convert them to single strands (so that the ‘probes’ can bind to them later).
B. Hybridisation: incubation of the nylon membrane with a single-stranded DNA probe. The probe will only bind to single-stranded DNA that contains the complementary nucleotide sequence of the STRs.
C. Gel electrophoresis: fragments of DNA are separated according to size. The sample of DNA fragments is placed in a well cut into an agarose gel and an electric current is applied. The smaller fragments of DNA pass more quickly through the gel towards the positive electrode (all the DNA fragments are negatively charged).
D. Extraction of DNA: shake a tissue sample with water-saturated phenol and chloroform at pH 7.8. Protein is precipitated and DNA enters the aqueous layer.
E. Visualisation: the probes bound to DNA bands on the nylon membrane are revealed using a chemical label.
F. DNA fragments created: bacterial restriction endonucleases (restriction enzymes) cut the long DNA molecules into fragments. Alternatively, the fragments are produced using the polymerase chain reaction (PCR). In DNA profiling these fragments contain unique non-coding regions of STRs.
G. Southern blotting: a thin nylon sheet is laid over the gel and blotting paper is laid on top. Buffer is drawn up by capillary action. The DNA fragments are dragged along and stick to the nylon membrane. In automated systems this step may not be required.