Rapid Pre-screening Analysis Through Heteroduplexes Analysis

Question 1

Specific point mutations may be demonstrated by ____ in addition to a number of other methods that may be appropriate, depending on whether ____ or ___ is being undertaken.

Answer 1

DNA sequencing; scanning for mutations; testing for a specific known mutation

Question 2

A ____ may be employed initially to identify a DNA segment, e.g., an exon, that may contain a mutation, prior to ___ of that region of the gene.

Answer 2

rapid screening strategy; DNA sequencing

Question 3

___ is a rapid screening strategy which is designed to detect properties of heteroduplex molecules such as ____ through a denaturing PAGE or ____ upon heating.

Answer 3

heteroduplex analysis; altered mobility; resistance to denaturation

Question 4

How do heteroduplexes form during heteroduplex analysis?

Answer 4

Base pairing between mutant and normal strands of DNA

Question 5

Heteroduplex form in the presence of DNA containing ____.

Answer 5

Heterozygous mutations

Question 6

During heterozygous analysis, the heteroduplex are allowed to form in the laboratory by heating the DNA to ____ and then cooling to ___.

Answer 6

95 degrees C; 25-40 degrees C

Question 7

____ is a rapid pre-screening strategy similar to the gel electrophoresis heteroduplex analysis but instead involves fluorescent detection of heteroduplexes, permitting automation and greater sensitivity.

Answer 7

conformation-sensitive capillary electrophoresis (CSCE)

Question 8

____ is a pre-screening strategy that detects the slight reduced ability of heteroduplex to bind to a DNA-binding cartridge

Answer 8

denaturing high-performance chromatography (dHPLC)

Question 9

This rapid pre-screening strategy is usually reserved for the repeated analysis of multiple samples.

Answer 9

denaturing-HPLC

Question 10

Advantages and disadvantages of dHPC

Answer 10

Advantages: highly sensitive;

Disadvantages: assays needs to be optimized for (1) the specific DNA sequence being tested and (2) subtle indications of the presence of mutation

Question 11

In this rapid pre-screening strategy, heteroduplexes are allowed to form, and then heated very gradually from around 65 to 95 degrees C to cause the strands to slowly separate. If heteroduplexes are present, the strands will separate slightly earlier indicating the possible presence of a mutation.

Answer 11

high-resolution melt curve analysis

Question 12

In high-resolution melt curve analysis, how is the presence of heteroduplex detected?

Answer 12

Detected as reduction in the fluorescence emitted by double-stranded DNA-binding dye molecules