Isolation Of DNA And Genes

Question 1

Why would we want to isolate DNA?

Answer 1

1. For genetic manipulations
2. For DNA analysis

Question 2

What are the steps for DNA isolation?

Answer 2

1. Cell lysis
2. DNA purification from the cell extract
3. Concentrate DNA
4. Measurements of DNA purity and concentration

Question 3

What do we not want in our sample of DNA?

Answer 3

• proteins
• ribosomes
• mtDNA
• lipids
• plasmids = only for miniprep

Question 4

What enzymes are used to disrupt different cell membranes?

Answer 4

Plants = cellulose

Bacteria = lysozyme

Eukaryotic cells = Sappanin

Question 5

How is DNA purified by using Phenol-Chloroform extraction?

Answer 5

1. Lysed cells/tissue are mixed with equal vol. of phenol-chloroform mixture.
2. Centrifuge
3. DNA concentration = .3M Sodium Acetate and 2.5 volumes Ethanol can be used to precipitate DNA from salt and sugar to concentrate it.

Question 6

How is DNA purified using commercial kits?

Answer 6

Column contains a silica membrane that binds DNA in the presence of a high concentration of salt

Silica binds DNA in a high concentration of salt
1.Lyse cells
2.Add high salt buffer
3.Wash with ethanol buffer
4.Elute with very low salt

Very quick, non-hazardous but more expensive and small volumes
Membrane can only bid a set amount of DNA

Question 7

What are restriction endonucelases and what do they do?

Answer 7

Molecular scissors that cut DNA at precise locations

Question 8

Why are restriction endonucleases used?

Answer 8

• to make recombinant DNA molecules = cloning
• to cut DNA into defined fragments = DNA fingerprinting and mutation analysis

Question 9

How do restriction enzymes cut DNA?

Answer 9

Make one cut in each of the sugar phosphate backbones of the double helix (breaks bond between 3’O and P) at their recognition site in the presence of Mg2+
-> Hydrolyses the phosphate group
-> Cut ends have a 5’ phosphate

Question 10

What does agarose gel electrophoresis do?

Answer 10

Separates DNA fragments

Question 11

What are the recognition sites for restriction enzymes?

Answer 11

Recognition sites for restriction enzymes are often palindromes (read the same backwards and forwards)

Question 12

How do samples enter the agarose gel?

Answer 12

Samples enter the gel and migrate according to charge, size and shape:

• DNA is negatively charged - migrates to positive electrode
• Smaller/compact molecules move more easily through gel than larger/long molecules