Lab 9 - Molecular Methods

Question 1

DNA isolation process:

Answer 1

1. Cell lysis to open cell
2. Adding detergent to remove lipid membrane
3. Remove proteins with proteases
4. Remove RNA with RNAase (if we want RNA have to remove DNA to get RNA sample)

Question 2

Methods

Answer 2

Phenol and chloroform (takes time)
DNA spin column based isolation
Protein binding with salts
DNA isolation by binding with a carrier

Question 3

Source of DNA

Answer 3

Peripheral blood
Hair
Sperm
Bones
Nails
Cheek epithelia
Urine

Question 4

RNA isolation

Answer 4

• Detection of RNA viruses and gene expression

- More unstable nuclei acid

Question 5

Sources of RNA

Answer 5

Peripheral blood

Fragments of tissue

Question 6

Restriction enzymes

Answer 6

• Bacterial enzymes also known as restriction endonucleases
• Found in bacteria, viruses and other prokaryotes
• Cleave human DNA into fragments at specific sequences, termed restriction sites

Question 7

Name of restriction enzymes

Answer 7

• Three-letter acronym for each enzymes derived from the source organism
• First letter from genus
• Next two letters represent species
• Additional letter or number represents the strain or serotypes
• The number tells which order of identification in the bacterium
• Ex. Hindll - Haemophilus influenza serotype d

Question 8

Restriction enzyme type 1

Answer 8

Complex, multiubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences.
Require ATP and S-adenosyl-L-methionine and Mg ions

Question 9

Restriction enzyme type 2

Answer 9

• Most common
• Cut DNA at defined positions close to or within their recognition sequences
• Do not need ATP but need S-adenosyl-L-methionine and Mg ions
• Type IIG restriction enzymes are large, combination restriction-and-modification enzymes. These enzymes cleave outside of their recognition sequences and can be classified as those that recognize continuous sequences

Question 10

Restriction enzyme type 3

Answer 10

• Large combination restriction-and-modification enzymes
• They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage

Question 11

Restriction enzyme type 4

Answer 11

-Recognise modified, methylated DNA

Question 12

Blunt ends

Answer 12

Straight cut. Both strands terminate in a base paire, and the ends ar always compatible

Question 13

Sticky ends

Answer 13

• A staggered cut generates two sticky ends
• A sticky or cohesive end has protruding single-stranded strands with unpaird nucleotides called overhangs
• Overhangs are in most cases palindromic