Recombinant DNA Techniques

Extracting DNA

Reasons to Collect DNA

-Genetic testing

-Body identification

-Analysis of forensic information

Outline and explain the purpose of: swabbing, lysis buffer, centrifugation, and isopropyl alcohol

-Swabbing is the process of collecting a sample of cells. An example of this is swabbing the inside of somebody’s mouth to collect cells for DNA extraction.

-The lysis buffer contains detergent and enzyme proteinase K and separates the DNA from the cell. The detergent breaks down the cell membrane and nuclear membrane so that the cells would burst open and release the DNA. The enzyme cuts down the histone proteins, which the DNA is wrapped around, and therefore frees the DNA.

-Centrifugation causes the remaining debris and protein to sink to the bottom of the test tube while the DNA remains distributed in the liquid. This thus separates the DNA from any debris and protein, which may interfere with analysis.

-Centrifugation can also be used after isopropyl alcohol is mixed with the DNA so that the DNA would sink to the bottom of the tube, allowing the DNA to be isolated.

-Isopropyl alcohol concentrates the DNA together so that it is visible to the naked eye. This is because DNA is not soluble in isopropyl alcohol.

Cutting DNA

Explain how enzymes cut DNA

-Enzymes that cut DNA are called restriction enzymes and they are mostly found in bacteria. These enzymes are sequence specific, which means that they only bind and cut specific DNA sequences. After the restriction enzymes bind to a specifically recognized sequence, they cut down the sugar phosphate backbones of the DNA strands.

Find and compare 3 different enzymes (one which must cut with ‘sticky ends’ and another with ‘blunt ends’)

-AluI (source: Arthrobacter luteus) produces blunt ends

-BamHI (source: Bacillus amyloliquefaciens) produces sticky ends

-EcoRI (source: Escherichia coli) produces sticky ends

-AluI, which produces blunt ends, cuts the strand symmetrically whereas BamHI and EcoRI, producing sticky ends, cut the strand asymmetrically. This means that BamHI and EcoRI create 5’ or 3’ overhangs on the DNA strand, which makes it easy to ligate. On the other hand, enzymes such as AluI leave no overhang because it cuts the strand symmetrically.

Separating nucleic acids/protein

Outline and explain steps: purpose of gel, electricity, ‘running buffer’, DNA standard, ‘loading buffer’, ethydium bromide

-The purpose of gel is to sort out DNA strand by filtering them with its sponge-like consistency.

-The electricity during electrophoresis makes the DNA strands in the wells of the gel move through the gel via a current. The smaller the strand, the further it will go in the gel.

-Running buffer is mixed with agarose when making the gel so that electrical charges can flow through the gel

-DNA standard contains DNA strands with known lengths; therefore it is used as a reference to compare the strands from the DNA sample with those from the standard.

-A loading buffer contains a dye that makes the DNA sample visible as it goes through the gel. Furthermore it makes the DNA sample thicker so that it will stay in the gel well instead of floating away.

-Ethydium bromide is used to stain the DNA sample because it binds to the DNA and is visible under fluorescent light.

Amplifying DNA

Outline and explain steps: role of primers, heating, cooling, reheating, over and over, compare amount of DNA at start to amount of DNA at finish.

-The primers in PCR attach to sites on the DNA strand that are at either end of the segment that is targeted to be copied.

-Heating the DNA causes it to unwind into two separate strands. Cooling it then leads to the primers in the solution to attach to the targets before the two DNA strands can rejoin. The solution gets heated again, causing the DNA polymerase to attach on a primer and add complementary nucleotides onto the strand. The temperature is then heated, cooled, and heated again to repeat this cycle, creating more complementary strands of the target sequence. After 4 cycles, there are 8 fragments that contain only the target sequence. After 30 cycles, there are over a billion fragments that contain only the target sequence and 60 copies of the longer length molecules.