Molecular Path Flashcards
What are the four applications of molecular techniques?
- Detection of inherited/acquired mutations
- Accurate diagnosis/classify tumor
- Detect infx agents
- Tissue typing, paternity testing
What are the advantages to molecular analysis over cytogenetic analysis?
Sensitive (genes are stable relative to proteins)
Not dependent on analysis of gene product
What are genome mutations?
The loss or gain of an entire chromosome
What are chromosome mutations?
Rearrangement of genetic material
What are gene mutations?
the complete deletion of a gene, or as little as a single point mutation
Where can gene mutations occur?
Introns, extrons
Triplet CAG repeat in an extron = ?
Huntingtons disease
CTG repeat in an UTR = ?
Myotonic dystrophy
What are the effects of mutations in promoters/enhancer regions of a DNA segment?
Increase or decrease in expression
Which are expressed, and which are cut out: exons and introns?
Exons = expressed Introns = cut out
What is the molecular basis of beta-thalassemia?
problem with the polyadenylation signal or in the promoter regions, causing a decreased expression
What is the direct detection of mutations?
DNA comparison with a known DNA sequence
What is an indirect detection?
Detection through linkage of the disease gene with a marker that is nearby
What are the three steps of direct detections of mutations?
- PCR amplification via primer
- Digest PCR products with appropriate endonuclease
- Separate digestion product with electrophoresis
What is allele-specific extension strategy?
For a known mutation, a primer is made before the mutated point. Then, marked nucleotides are added. Since point mutation is known, then the markers will indicate whether it is a wild, mutated, or mixed gene.
What is allele-specific extension strategy?
For a known mutation, a primer is made up until the mutated point. Then, marked nucleotides are added. Since point mutation is known, then the markers will indicate whether it is a wild, mutated, or mixed gene.
How are linkage analysis analyzed?
Endonuclease used to cut marker gene
Southern blot will display different cut genes of the specific gene in question (d/t the use of a probe)
What is the difference between linkage analysis and detection of genetic mutations through endonucleases?
A specific probe is used to only look at the mutated linker gene in question, rather than run all of the DNA in an electrophoresis
What is the use of genetic techniques for HIV and Hep C?
Monitor Viral load via [RNA]
What is the difference between linkage analysis and detection of genetic mutations through endonucleases?
A specific probe is used to only look at the mutated linker gene in question, rather than run all of the DNA in an eletrophoresis
What is indirect detection of a gene?
Through linkage of the disease gene with a marker that is nearby
What are the advantages of indirect detection/linkage analysis?
Does not require gene sequence to be known
Does require that polymorphism is sufficiently close to the mutation
How are linkage analysis analyzed?
Endonuclease used to cut marker gene
Southern blot will display different cut genes
What is spectral karyotyping (SKY)? What is this technique used for?
Painting all chromosomes a specific color, looking for translocations
What is the use of genetic techniques for HIV?
Monitor Viral load
What is PCR used for?
Detect type of infectious agent.
What are the characteristics of a gene that can be used to identify an individual?
Must by highly polymorphic (like MHC)
What is the purpose of a ladder in DNA analysis?
the ladder is a control DNA segment for DNA size
Can you totally exclude/include suspects with DNA forensic analysis? Why or why not?
Can totally exclude
Cannot totally include b/c MHC not completely unique
FISH =?
Staining DNA and looking for abnormal numbers
What is spectral karyotyping?
Painting all chromosomes a specific color, looking for translocations
What is the array-based comparative genomic hybridization?
Color labeled normal and abnormal genes hybridized to sample. ***
What is aneuploidy?
Lack of a chromosome (like Down)
What causes Beta-thalassemia?
Point mutation in the promoter/enhancer changes gene expression
Changes in the 5’ cap
Is PCR direct or indirect technique of detecting genetic mutations?
Direct
What are the two major advantages of molecular analysis over karyotyping?
- Sensitivity
2. Not gene product dependent–any cell works
CF is caused by what genetic mutation?
Frameshift mutation
What is RFLP (restriction fragment length polymorphism)?
Mutation causes addition or subtraction of an endonuclease site. Therefore, different gene fragment lengths are produced
What is the principle behind RFLP (restriction fragment length polymorphism)?
Mutation causes addition or subtraction of an endonuclease site. Therefore, different gene fragment lengths are produced
What is the method of detection of RFLP?
Southern blot of amplified sample gene, compared to a normal gene (must know normal)
What is the difference between direct and indirect detection of RFLP?
Direct = it is the loss of the endonuclease site that causes the disease
Indirect = only correlated
What are the advantages of indirect detection RFLP?
Advantage = do not need to know specific gene, just closely associated one
CGG repeat on an X chromosome= ?
Fragile X sydrome
GAA repeat in an intron = ?
Friedreich ataxia
A mutation in the 3’ polyadenylation signal may lead to what?
Unstable mRNA = not expressed protein, or decreased expression
How does forensic medicine work?
Run MHC genes of suspected persons with evidence, and compare
Where do epigenetic changes usually occur?
at promoters at the CpG islands
Histone deacetylation is associated with an increase or a decrease in gene expression?
Decrease
What is array-based comparative genomic hybridization?
Tested DNA and reference DNA are labeled with two fluorescent dyes and hybridized to slide spotted with DNA probes. This can detect amplification or deletion of a gene, depending on the color at each probe.
