Molecular pathology

Question 1

genome mutations

Answer 1

• loss or gain of an entire chromosome (cytogenetic or molecular anylaysis)

Question 2

Chromosome mutations

Answer 2

rearrangement of genetic material (cytogenetic or molecular analysis)

Question 3

Gene mutation

Answer 3

• the complete deletion of a gene or as little as a single point mutation
• they are submicroscopic and can be detected ONLY with molecular techniques
• Molecular analysis is NOT dependent on analysis of the gene product and so almost any cell will work

Question 4

What are the types of gene mutations

Answer 4

• point mutations
• insertion
• deletions
• trinucleotide repeat mutation
• • could be in the RNA coding and non-coding regions such as enhancers, promoters or introns**

Question 5

what are the types of DNA diagnosis

*

Answer 5

• Direct detection = of mutation in the DNA by comparison with a known DNA sequence
• INDIRECT detection = thought linkage of the disease gene with a marker that is nearby (detect something else)

Question 6

Direct Detection of mutations

Answer 6

• If a mutation alters or destroys a restriction endonuclease site on the DNA then it can be detected by:
• -> amplifying the region of interest with PCR
• -> digesting the PCR products with appropriate restriction enzyme
• -> separating the digestion products by electrophoresis
• DIRECT DETECTION

Question 7

allele specific extension strategy

Answer 7

• can identify mutations at a specific nucleotide position
• can be used in so called “real time” during the early phase of DNA amplification in PCR so the time required for analysis is reduced
• detects the presence of mutant DNA in mixtures by tagging nucleotides with florescent
• DIRECT DETECTION

Question 8

INDIRECT detection of mutations

Answer 8

• indirect detection though linkage of the disease gene with a marker that is nearby
• can be accomplished through site and length polymorphisms
• does NOT require the gene sequence to be known
• DOES REQUIRE the polymorphism used for detection is sufficiently close to the mutated gene so that the marker and mutation are INHERITED TOGETHER

Question 9

Direct Detection of mutations

*

Answer 9

• If a mutation alters or destroys a restriction endonuclease site on the DNA then it can be detected by:
• -> amplifying the region of interest with PCR
• -> digesting the PCR products with appropriate restriction enzyme
• -> separating the digestion products by electrophoresis
• DIRECT DETECTION

Question 10

allele specific extension strategy

*

Answer 10

• can identify mutations at a specific nucleotide position
• can be used in so called “real time” during the early phase of DNA amplification in PCR so the time required for analysis is reduced
• detects the presence of mutant DNA in mixtures by tagging nucleotides with florescent
• DIRECT DETECTION

Question 11

INDIRECT detection of mutations

*

Answer 11

• indirect detection though linkage of the disease gene with a marker that is nearby
• can be accomplished through site and length polymorphisms
• does NOT require the gene sequence to be known
• DOES REQUIRE the polymorphism used for detection is sufficiently close to the mutated gene so that the marker and mutation are INHERITED TOGETHER

Question 12

Epigenetic alterations

Answer 12

• Increase methylation of DNA (usually promoters of CpG) leads to DECREASE in the expression of gene
• HISTONE deacetylation is associated with DECREASE in gene expression

Question 13

Array-based comparative genomic hybridization (array CGH)

Answer 13

• Test genomic DNA and a reference DNA (control) are labeled with two different flourescent dyes and hybridized to slide spotted with DNA probes that span the human genome
• Yellow denotes equal amounts of BOTH DNA’s but if there is amplification or deletion of DNA from the test sample then the spot will be green or red