Molecular Genetic Techniques

Question 1

What are restriction endonucleases and what do they do?

Answer 1

They are DNA cutting enzymes that are produced by bacteria and are used to cleave DNA to produce restriction fragments

Question 2

In what organisms are restriction enzymes normally found?

Answer 2

Bacteria

Question 3

How are restriction enzymes named?

Answer 3

After the Bacteria it comes from

Question 4

What is the purpose of the lacZ gene in the pUC18 plasmid for cloning DNA segments?

Answer 4

It allows for detection of colonies having plasmid with insert by turning them blue

Question 5

What is the purpose of the ampicillin resistance gene in the pUC18 plasmid for cloning

Answer 5

It makes it so only bacteria with the plasmid will grow

Question 6

What is the purpose of gel electrophoresis?

Answer 6

It is used to separate molecules on the basis of their size and charge

Question 7

Toward which electrode (positive or negative) does DNA travel during gel electrophoresis?

Answer 7

Positive

Question 8

After gel electrophoresis, are the DNA molecules at the bottom of the gel small or large?

Answer 8

Small

Question 9

What is the purpose of polymerase chain reaction (PCR)?

Answer 9

In vitro amplification of DNA

A way to copy a specific sequence of DNA without vectors and
host cells (alternative to cloning)

Question 10

What is special about Taq polymerase?

Answer 10

The Taq polymerase is a DNA polymerase isolated from the bacterium Thermus aquaticus

This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases

Question 11

What are the three steps in each cycle of a PCR reaction, and what happens during each step?

Answer 11

Step 1: DNA is heated to 90°C - 100°C to separate the two strands of DNA

Step 2: The DNA is quickly cooled to 30°C - 65°C to allow short single-stranded primers to anneal to their complimentary sequences

Step 3: The solution is heated to 60 C – 70 C; DNA polymerase synthesizes new DNA strands, creating two new, double-stranded DNA molecules.

Question 12

Dideoxy sequencing) relies on the use of ddNTPs. How are ddNTPs different from dNTPs?

Answer 12

They are a deoxynucleotide with a hydrogen at 3’ instead of an OH

Question 13

What happens when the polymerase enzyme incorporates a ddNTP during DNA synthesis?

Answer 13

It causes DNA synthesis to terminate