06: Laboratory Methods for Genetic Mutation Analysis

Question 1

What is a restriction site?

Answer 1

Location on a DNA molecule containing specific (4-8 base pairs in length)sequences of nucleotides, which are recognized by restriction enzymes; generally palindromic sequences.

Question 2

Describe for PCR:

1. Mechanism
2. Use
3. Restrictions

Answer 2

1. Creates identical copies of a section of DNA.
2. Look for polymorphisms (SNPS, microsatellites, variable number tandem repeats [VNTRs]), insertions and deletions; known micro mutations.
3. Qualitative tests, so heterozygous deletions not detected in primer site.

Note: Used in forensics for genetic fingerprinting.

Question 3

Describe for Multiple Ligation Probe Amplification:

1. Mechanism
2. Use
3. Restrictions

Answer 3

1. Quantifies a gene
2. Detects deletions in heterozygotes; known micro mutation
3. No other use

Question 4

Describe for Southern Blot:

1. Mechanism
2. Use
3. Restrictions

Answer 4

1. Cut DNA at restriction sites to get predictable fragment pattern. Use fluorescent probe to bind complementary sequences. Variations/abnormalities involving restriction sites are detected.
2. Large scale deletions (>100bp); macro known mutation
3. Limited resolution; cannot detect <100bp.

Question 5

Describe for genome sequencing:

1. Mechanism
2. Use
3. Restrictions

Answer 5

1. Cuts genome randomly into small pieces that overlap; computre uses the overlap to put the pieces in order.
2. Determine sequence; unknown micro.
3. Microsatellites and copy number variation not reliably reported.

Question 6

Describe for genome-wide association studies:

1. Mechanism
2. Use
3. Restrictions

Answer 6

1. Genome sequence large number of people and try to correlate a disease with a genetic variation using a Manhattan plot.
2. Finds genes that cause disease.
3. Statistically significant relations may not be clinically useful; may identify marker travelling in linkage disequilibrium and not the causative gene itself.

Question 7

Describe for comparative genomic hybridization:

1. Mechanism
2. Use
3. Restrictions

Answer 7

1. Compares genome of sick vs. healthy individual
2. Detects changes at the chromosomal level, such as copy number variation (CNV); unknown macro
3. Cannot detect changes <1Mb, cannot accurately report deletions/duplications above 3-4.

Question 8

Describe for microarrays:

1. Mechanism
2. Use
3. Restrictions

Answer 8

1. Plate has many different gene sequences attached. Take an mRNA from two samples and hybridize them with the plate and compare their expression levels using a heat map.
2. Gene expression profiling in cancer; RNA level of expression in cancer
3. Test destroys architecture of cell; cannot determine if expression is just of tumor cells or other cells too.

Question 9

Describe for RNA in situ hybridization:

1. Mechanism
2. Use
3. Restrictions

Answer 9

1. Stains for mRNA in a tissue
2. See gene expression while keeping cellular architecture intact; RNA level of expression in cancer
3. Can only do a few genes at a time

Question 10

Describe for immunocytochemistry:

1. Mechanism
2. Use
3. Restriction

Answer 10

1. Stains for protein in the tissue
2. See protein expression while keeping cellular architecture intact; protein level of expression
3. Can’t detect the size of the protein like you can with a western blot (immunoblot).