MDSC 515 FINAL EXAMS ACTUAL QUESTIONS
AND ANSWERS SURE A+
✔✔Met kinase and exon skipping - ✔✔-MET is a receptor tyrosine kinase containing
regulatory exon 14 that controls protein stability
-exon skipping mutations in lung cancer remove exon 14 - MET is no longer regulated
-MET is over expressed and acts as an oncogene (proliferation)
✔✔Consequences of translocations - ✔✔-break large intronic regions
-fusion mRNA can be formed that forms an abnormal protein if transaction places part
of one gene next to another then fusion can occur during transcription
-there could also be a promoter-gene fusion causing altered expression
✔✔differences between RNA and DNA - ✔✔-single stranded vs double stranded
-no introns
-uracil replaces thymine
-additional 2' hydroxyl group on ribose sugar
✔✔pros of analyzing DNA - ✔✔-stable, abundant
-equal numbers of all genes (2 per cell)
-intrinsic copy number controls
-somatic and inherited mutations are easy to test for
-antisense strand confirmation of mutations
, -easier to get from tissue
✔✔cons of analyzing DNA - ✔✔-intronic distance limits gene fusion detection by PCR
-no expression information (geno=pheno correlation)
-pseudogene amplification
✔✔pros of analyzing DNA - ✔✔-more direct gene-pheno correlation - can use levels to
see if they respond to therapy - proteins are linked to phenotype
-lack of introns facilitates fusion gene detection
-expression level analysis - if RNA expressed then protein expressed
✔✔cons of analyzing RNA - ✔✔-stability - must be protected and processed within
minutes and very carefully
-splice variants - several may not be an accurate biomarker
-difficult to detect low level gene expression
-difficult to get from tissue
-RNA alteration may not be heritable
-detection of low expression RNA can be easily obscured by contaminating normal
tissue
✔✔PCR steps - ✔✔1. denaturation
2. annealing
3. extension
✔✔critical components of PCR - ✔✔-target: DNA or gene to be amplified
-primers: specific to the target - typically 18-20bp is sufficient to be unique in the human
genome
-nucleotides
-polymerase: typically taq-polymerase
-magnesium: divalent cations required for nucleic acid modifying enzymes, too low-low
amplification or too high non-specific amplification
-buffer
✔✔endpoint PCR - ✔✔-all PCR's hit a set plateau level - end-point
-at endpoint, there is no relationship between starting DNA amount and PCR amount
-can only have binary detection but can detect in/dels based on size (cannot find the
small ones)
-oncogenes not likely detected as PCR is not specific enough for the middle regions
✔✔allele specific PCR-detecting SNV's - ✔✔-primer stability depends on 3' clamping -
no binding on 3' = no extension by polymerase
-primers designed against a 3' SNP or point mutation can generate a specific PCR
fragment even if different by one nucleotide - allele specific
-one primer is set for mutant allele and one is set for the normal allele - amplification of
the mutant allele indicates that a patient harbours the point mutation
AND ANSWERS SURE A+
✔✔Met kinase and exon skipping - ✔✔-MET is a receptor tyrosine kinase containing
regulatory exon 14 that controls protein stability
-exon skipping mutations in lung cancer remove exon 14 - MET is no longer regulated
-MET is over expressed and acts as an oncogene (proliferation)
✔✔Consequences of translocations - ✔✔-break large intronic regions
-fusion mRNA can be formed that forms an abnormal protein if transaction places part
of one gene next to another then fusion can occur during transcription
-there could also be a promoter-gene fusion causing altered expression
✔✔differences between RNA and DNA - ✔✔-single stranded vs double stranded
-no introns
-uracil replaces thymine
-additional 2' hydroxyl group on ribose sugar
✔✔pros of analyzing DNA - ✔✔-stable, abundant
-equal numbers of all genes (2 per cell)
-intrinsic copy number controls
-somatic and inherited mutations are easy to test for
-antisense strand confirmation of mutations
, -easier to get from tissue
✔✔cons of analyzing DNA - ✔✔-intronic distance limits gene fusion detection by PCR
-no expression information (geno=pheno correlation)
-pseudogene amplification
✔✔pros of analyzing DNA - ✔✔-more direct gene-pheno correlation - can use levels to
see if they respond to therapy - proteins are linked to phenotype
-lack of introns facilitates fusion gene detection
-expression level analysis - if RNA expressed then protein expressed
✔✔cons of analyzing RNA - ✔✔-stability - must be protected and processed within
minutes and very carefully
-splice variants - several may not be an accurate biomarker
-difficult to detect low level gene expression
-difficult to get from tissue
-RNA alteration may not be heritable
-detection of low expression RNA can be easily obscured by contaminating normal
tissue
✔✔PCR steps - ✔✔1. denaturation
2. annealing
3. extension
✔✔critical components of PCR - ✔✔-target: DNA or gene to be amplified
-primers: specific to the target - typically 18-20bp is sufficient to be unique in the human
genome
-nucleotides
-polymerase: typically taq-polymerase
-magnesium: divalent cations required for nucleic acid modifying enzymes, too low-low
amplification or too high non-specific amplification
-buffer
✔✔endpoint PCR - ✔✔-all PCR's hit a set plateau level - end-point
-at endpoint, there is no relationship between starting DNA amount and PCR amount
-can only have binary detection but can detect in/dels based on size (cannot find the
small ones)
-oncogenes not likely detected as PCR is not specific enough for the middle regions
✔✔allele specific PCR-detecting SNV's - ✔✔-primer stability depends on 3' clamping -
no binding on 3' = no extension by polymerase
-primers designed against a 3' SNP or point mutation can generate a specific PCR
fragment even if different by one nucleotide - allele specific
-one primer is set for mutant allele and one is set for the normal allele - amplification of
the mutant allele indicates that a patient harbours the point mutation
