Tools of Molecular Biology

Question 1

What are three palindromic double-stranded DNA sequences that are likely to be cut by restriction endonucleases?

Answer 1

1. (5')GGATCC(3')
   (3')CCTAGG(5')
2. (5')GATC(3')
    (3')CTAG(5')
3. (5')TTACGG(3')
    (3')AATGCC(5')

Question 2

Gel electrophoresis separates DNA based on what characteristic?

Answer 2

Size

Question 3

In gel electrophoresis, which node does the DNA move towards?

Answer 3

Anode (positive electrode).

Question 4

In gel electrophoresis, what is the relationship with the rate at which DNA moves and size.

Answer 4

Inverse. The bigger the size, the slower the rate.

Question 5

How are DNA fragments visualized in gel electrophoresis?

Answer 5

Dyed with fluorescent dye and radioactive probe.

Question 6

What is Restriction Fragment Length Polymorphisms (RFLP)?

Answer 6

Genetic variations among individuals that affect recognition sites, and therefore, lead to variations in sizes of DNA fragments when digested by a particular enzyme. Can be compared to wild type if suspect mutation.

Question 7

How is RFLP used to detect the HbS mutation in sickle cell anemia?

Answer 7

Because the HbS mutation destroys one of the restriction sites on the Beta-globin gene (typically digested by restriction enzyme MstII), the DNA fragment is longer than the wild type fragment after digestion. Based on this difference in size mutant gene can be separated via gel electrophoresis.

Question 8

**How is RFLP utilized in DNA Fingerprinting?

Answer 8

.

Question 9

What are the three experimental stages in diagnosing with RFLP?

Answer 9

1. Cleavage by restriction enzyme.
2. Gel Electrophoresis
3. Southern blotting

Question 10

What are the three experimental stages in DNA fingerprinting?

Answer 10

1. PCR
2. Electrophoresis
3. Southern blot

Question 11

What are the transfer techniques of DNA, RNA, and protein?

Answer 11

DNA: Southern blot
RNA: Northern blot
Protein: Western blot

Question 12

**What are three characteristics of a hybridization probe used to detect a specific DNA sequence on a membrane?

Answer 12

1. Length: length will determine temperature at which annealing of DNA can occur.
2. Sequence: probe must be complimentary
3. Radioactivity or Dye: to help visualize

Question 13

What enzyme is responsible for copying a DNA sequence into a DNA sequence for recombination?

Answer 13

DNA polymerases

Question 14

What enzyme is responsible for copying a RNA sequence into a DNA sequence for recombination?

Answer 14

Reverse transcriptase

Question 15

What enzyme is responsible for joining DNA fragments for recombination?

Answer 15

DNA ligase (forming phosphodiester bonds between 5’ end and 3’ end.)

Question 16

How are microarrays used to measure levels of mRNA (gene expression?

Answer 16

cDNA obtained from mRNA will bind to DNA on microarray if complimentary. If complimentary, can be visualized to mark level presence.

Question 17

List the types of cloning vectors.

Answer 17

Plasmid, bacteriophage, cosmid, bacterial artificial chromosome, yeast artificial chromosome, retrovirus.

Question 18

Plasmid

Answer 18

Circular DNA, easy to clone, but slow, and passive uptake by cells, E. coli is host

Question 19

Bacteriophage

Answer 19

Much more efficient than plasmids due to active uptake by cells, E.coli is host

Question 20

Cosmid

Answer 20

Bacteriophage/plasmid hybrid

Question 21

Yeast artificial chromosome

Answer 21

Also used for cloning long segments of DNA

Question 22

Retrovirus

Answer 22

contains RNA that is reverse transcribed in host