12.2 Molecular Techniques

Question 1

What are restriction enzymes?

Answer 1

Endonuclease enzymes produced by bacteria. They cut double stranded DNA at specific sites called restriction sites. These restriction sites are 4-8 bp long and they are palindromic. the cut in made into the sugar-phosphate backbone of each strand. When they cut, they produce sticky ends, staggered cuts.
restriction enzymes are used to isolate a gene/DNA segment of interest.

Question 2

What recognizes and cuts specific palindromic sequences?

Answer 2

Restriction enzymes

Question 3

What sort of cut results from restriction enzmye activity?

Answer 3

Double strand cut. The cuts are not clean but staggered, leaving overhangs or “sticky ends”.

Question 4

What do restriction enzymes recognize?

Answer 4

They recognize specific palindromic DNA sequences of 4-8 bp long.

Question 5

What sort of enzymatic activity do restriction enzymes have?

Answer 5

endonuclease

Question 6

What is a recognition site?

Answer 6

A recognition site in the specific palindromic DNA sequence (4-8bp) that restriction enzmyes recognize and cut.

Question 7

What sort of organism produces restriction enzymes?

Answer 7

Bacteria

Question 8

On which basis are DNA fragment separated in DNA electrophoresis?

Answer 8

Size, molecular weight,

Question 9

Why do fragments migrate in electrophoresis, and in which direction?

Answer 9

The migrate because these DNA protein fragments are negatively charged. Their phosphate groups on the outside (part of the backbone) make them highly negatively charged. We will induce a current (electric field) running through the gel, and to the negatively charged fragments will migrate towards the + electrode (= positively charged anode).

Question 10

What are the requirements for gel electrophoresis?

Answer 10

1. Gel
2. Buffer
3. Power supply
4. Staining chemical

Question 11

What are plasmids?

Answer 11

Small circular double-stranded DNA molecules. They are found in bacteria. They have the ability to self replicate in bacterial milieu.

Question 12

What are plasmids used for in molecular biology?

Answer 12

They are used for cloning genes of interest. We insert fragments with sticky ends resulting from restriction into the plasmid, and then when the plasmid replicates, it will also replicate our gene.

Question 13

What therapeutic interest is there for cloning human genes?

Answer 13

We can clone genes coding for insulin for example.
Or genes coding for CFTR for CF patients.
Or SERCA for heart failure.

Question 14

Why is a reverse transcriptase necessary for therapeutic cloning of insulin for example?

Answer 14

Because if we use DNA clone as it is, it contains introns! So what we actually do is use an mRNA molecule, and then a reverse transcriptase (in some viruses) which will in turn give us DNA. The template is no longer DNA that we insert in plasmid but mRNA.

Question 15

Which method is used to amplify a target DNA sequence?

Answer 15

PCR, polymerase chain reaction.

Question 16

What are the steps in DNA cloning?

Answer 16

1. Isolate relevant gene of interest following digestion by restriction enzyme
2. Insert gene of interest into plasmid vector = recombinant DNA molecule
3. Introduce recombinant DNA molecule into suitable host cells
4. Identify and isolate the clone containing the DNA of interest

Question 17

How does PCR work (steps)?

Answer 17

PCR = polymerase chain reaction
We use heat to break H bonds between the strands. They separate.
We insert 2 DNA primers into the tube. The primers define the region to be copied.
We cool down slightly the tube, enabling H bonds to reform, but not with their complementary strand but with complementary primer! Now basepairing occurs from primer segment along the single strand by thermostable DNA polymerase Taq.
After this process, we have 2 double stranded segments with our gene of interest. then we do it again, and again, making exponential copies of the gene.

Question 18

In which molecular investigation technique is a thermostable DNA polymerase involved?

Answer 18

PCR, the DNA polymerase will synthesize the complementary strand starting from the primers.

Question 19

What do the 2 primers do in PCR?

Answer 19

They take the place of the complementary DNA strand after heating and recooling. then tehy enable DNA polymerase to bind and start synthesis.

Question 20

What is SDS-PAGE, what does it separate and on what basis?

Answer 20

SDS-PAGE is a protein separation method. they will be separated on the basis of size. The method is analogous to that of electrophoresis, but with proteins instead of DNA segments.
SDS is sodium dodecyl sulfate, it is an anionic detergent that linearizes proteins and charges the proteins negatively to ensure their migration.