Molecular biology tools

Question 1

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

Answer 1

AATT, GGATCC, AAGCTT, GATC

Question 2

Explain the principle of electrophoretic separation

Answer 2

DNA molecules are separated on the basis of their size. you have an agarose gel and apply an electric current. DNA Moves towards positive charge. Larger the segment of DNA, the slower it moves.

Question 3

Give an example of a disease that can be diagnosed using a restriction fragment length polymorphism (RFLP) and a use of DNA fingerprinting

Answer 3

An HbS mutation, causing sickle cell anemia can be detected using RFLP and DNA fingerprinting.

Question 4

Describe at least three experimental stages required in RFLP and DNA fingerprinting

Answer 4

1. digest their genomic DNA with MstII, a restriction enzyme
2. run out the DNA on an electrophoresis gel,
3. probe with a region spanning the MstII site by southern blotting. if you get a large fragment, its a positive test for sickle cell because MstII cannot cleave when the A to G mutation in HbS occurs

Question 5

List the names given to the transfer of DNA, RNA and protein respectively from an electrophoresis gel to a membrane.

Answer 5

Transfer of DNA is a southern Blot
Transfer of RNA is a northern Blot
Transfer of Protein is a western Blot

Question 6

Describe three characteristics of a hybridization probe that you will use to detect a specific DNA sequence on a membrane.

Answer 6

1. It must be complementary to the specific sequence that you are trying to detect,
2. it must be attached to a labeled probe,
3. It must be a single stranded DNA molecule

Question 7

Recall the classes of enzymes that are used in recombinant technology to: a) copy a DNA sequence into a DNA sequence, b) copy an RNA sequence into a DNA sequence, c) join DNA fragments.

Answer 7

1. DNA polymerase
2. Reverse transcriptase
3. DNA ligase

Question 8

Describe the three main stages that are repeated during PCR amplification. State the approximate temperature of each step, and relate this temperature to the state of the DNA molecules in the PCR reaction.

Answer 8

1. DNA template is heated to 94 dC and DNA is denatured
2. The primers (or oligos) anneal or hybribize with complementary sequences in the template. at 50-60 dC
3. elongation from the primers at 72 dC by Taq polymerase

Question 9

Describe at least 1 distinct use for PCR amplification in the diagnosis of a genetic condition in your patients

Answer 9

cystic fibrosis: The most frequently occurring mutation is deletion of phenylalanine 508 delta-F508. Although only missing one amino acid, it is identified as non-functional and does not undergo the usual final modifications within the endoplasmic reticulum. mutant protein is degraded.
Primers are designed such that the nucleotide(s) distinguishing the mutant and wild type alleles is at the 3’ terminus of the primer.

Tay-Sachs disease, Beta-thalassaemia, HLA typing are other examples.

Question 10

Compare and contrast the molecular details of the processes of DNA sequencing and PCR amplification in a short paragraph.

Answer 10

TBA

Question 11

Be aware of the different types of cloning vectors and their general features

Answer 11

TBA

Question 12

Describe the use of microarrays for measuring levels of mRNA (e.g. gene expression)

Answer 12

TBA