Tools of Molecular Biology

Question 1

Name 4 methods of characterizing a piece of DNA and when they are generally used.

Answer 1

1. Restriction Digestion Analysis: quick and cheap way to see if DNA segment of interest was inserted
2. Hybridization: when you have many clones to screen
3. DNA Sequencing: when trying to identify a mutated recombinant clone => determination of a complement strand sequence of the unknown strand
4. PCR Amplification: when many copies of a gene are needed => creates 2^n copies; n=# of cycles

Question 2

Give an example of a disease that can be diagnosed using a restriction fragment length polymorphism.

Answer 2

Screening for the HbS mutation of sickle cell anemia:
HbS mutation destroys restriction site for endonuclease MstII
=> Perform restriction digest with diagnostic enzyme
=> Southern blot or PCR analysis
=> electrophoresis to determine size of restriction fragment

Question 3

Name the three characteristics of a hybridization probe that is needed to detect a specific DNA sequence on a membrane.

Answer 3

Radioactive
Single-stranded
Complementary to DNA sequence of interest

Question 4

When are each of the three blotting techniques used?

Answer 4

1. Southern blotting detects specific DNA fragments from complex mixtures via hybridization
2. Norther blotting identifies RNA fragments from a complex mixture of DNA fragments
3. Western blotting identifies proteins from complex mixtures using antibodies

Question 5

Which class of enzyme is used to copy a DNA sequence into a DNA sequence?

Answer 5

DNA polymerase

Question 6

Which class of enzyme is used to copy an RNA sequence into a DNA sequence?

Answer 6

Reverse transcriptase

Question 7

Which class of enzyme is used to join DNA fragments?

Answer 7

DNA ligase

Question 8

What are the three main stages that are repeated during PCR amplification?

Answer 8

1. DNA strands are denatured @ 95*C
2. Primers hybridize to the template strand @ 55*C
3. DNA polymerase copies each strand by extending the 3’ end of the primer @ 72*C

Question 9

What are the components necessary to perform PCR amplification?

Answer 9

Thermally stable DNA polymerase (Taq)

Four nucleotide triphosphates, containing primers and DNA template

Question 10

How is PCR amplification used to diagnose a disease?

Answer 10

By using PCR primers that will only hybridize with a mutant allele versus the normal allele, therefore the presence of a PCR product indicates the presence of the disease. (Can test multiple disease at the same time)

Question 11

Name 4 disease that can be diagnosed via PCR amplification.

Answer 11

Cystic fibrosis
Tay-Sachs disease
beta-Thalassaemia
HLA typing

Question 12

What is the main similarity and difference between PCR amplification and DNA sequencing?

Answer 12

Similarity: both use primer sequence to initiate replication of genes

Difference: PCR works on double-stranded DNA; DNA sequencing works on a single-stranded DNA fragment using ddNTPs (and PCR does not)