Explain the reasons for the key steps involved in DNA extraction, PCR and electrophoresis below.
Key steps -
1. Collect DNA Evidence from crime scene.
2. Extract DNA.
3. Use restriction enzymes to cut the DNA into different sized fragments.
4. Amplify DNA sample using PCR.
5. Seperate the fragments using electrophoresis into bands of DNA.
6. Make the DNA band visible.
7. Compare the position of the DNA bands with suspect samples.
These are the basic steps in identifying or excluding suspects in a crime. Each step has to be done carefully, as mistakes can be made in each--and we are talking about someone being found guilty or not guilty of a serious crime.
1. Collect DNA Evidence from crime scene--must be done carefully, not contaminated w/other DNA, must be properly catalogued.
2. Extract DNA--DNA must be extracted carefully from the cells; ie, the cells are disrupted, and DNA separated from other cell components via use of chelating agents and centrifuge.
3. Use restriction enzymes to cut the DNA into different sized fragments-These must be chosen carefully in order to obtain similar segments of all crime scene DNA, obtaining sections of the DNA likely to hold differences between people.
4. Amplify DNA sample using PCR--there is frequently not a great deal of DNA available. After desired segments are "cut" with restriction enzymes, PCR (polymerase chain reaction) is used to make ~1 billion copies of the DNA segment in a couple of hours. The sample is heated to split the double strands; the temp is lowered, allowing primers and TAQ polymerase to anneal (hook on) bases to the strands; the cycle is repeated, doubling the number of segments each time.
5. Seperate the fragments using electrophoresis into bands of DNA--a small amount of the DNA is loaded into the well of a gel, and elecricity is applied so that the negatively charged DNA fragments migrate to the positive side. The smaller the fragment, the more quickly (and therefore further) it travels through the gel.
6. Make the DNA band visible--can either be done by staining the gel after electrophoresis, or by adding a stain to the gel before it is cast so that the bands show up in UV light.
7. Compare the position of the DNA bands with suspect samples--if the crime scene DNA does not match the subject, s/he can be excluded. If it does, tests on other sections will be run to make sure they also match.