MDSC 515 FINAL PAPER 2025/2026 QUESTIONS AND
SOLUTIONS GRADED A+ TIP
✔✔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
-mutant primer won't bind to the normal strand but wild type will always show up on the
mutant DNA
✔✔reverse transcription PCR - ✔✔-RNA cannot be used directly for PCR
-cDNA synthesis required to perform traditional PCR
-one step: reverse transcription (RNA to DNA) and PCR in one tube
-two step: cDNA synthesis first and then used as a target for separate PCR reaction
-allows detection of RNA based alterations such as exon skipping and RNA fusions
✔✔exon skipping from reverse transcription PCR - ✔✔-PCR primers designed across
the skipped exon show a size difference in comparison to end point PCR - missing a
section so they are smaller
✔✔RNA fusions from reverse transcription PCR - ✔✔-PCR primers designed to the two
different exons will normally not amplify but if the fusion product is present then the PCR
will amplify the abnormal mRNA
✔✔cytogenetics - ✔✔study of the strutter and function of chromosomes and their role in
human disease
-resolution of 5-10 mb
-highly subjective (different people see different things)
, -requires cell culture - cannot just be from tissues
-diagnostic yield is low
-good to test for aneuplodies but not other structural variants
✔✔constitutional cytogenetics - ✔✔diagnosis of heritable (germline) genetic
abnormalities in the prenatal, paediatric and adult settings
✔✔cancer cytogenetics - ✔✔detection of acquired (somatic) genetic abnormalities for
the diagnosis, prognosis, therapy, and/or monitoring for many types of cancer
✔✔variation in chromosome number and structure - ✔✔-aneuploidy and policy changes
- from errors occurring during meiotic or mitotic segregation
-translocation, inversions, insertions, deletions, duplications
✔✔balanced structural chromosome changes - ✔✔-no loss/gain of genetic material
-translocations
-inversions
-insertions
✔✔unbalanced structural chromosome changes - ✔✔-loss/gain of genetic material
-deletions
-duplications
✔✔paracentric vs pericentric inversion - ✔✔-para involves the centromere and peri
doesn't
-if the region inverted loops when the centromere is involved and there is recombination
then there will be loss or gain in the offspring
✔✔isodicentric chromosome - ✔✔-mirror image duplication involving the centromere
✔✔isochromosome - ✔✔-mirror image duplication not involving the centromere
✔✔ring chromosomes - ✔✔-when the chromosome is fractured and it tries to fix itself
-chromosome breaks in two places and the ends fuse together
✔✔chromosomal SNP microarray detection - ✔✔-genomic imbalances
-duplications and deletions (copy number loss)
-whole chromosomal aneuploidies
-triploidy (69 chromosomes)
-microdeletions and microduplications - CNV's and unbalanced rearrangements - loss of
heterozygosity - more related = more haplotypes the same
-mosaic imbalances >10% - when different cells in the body have different genotypes
-regions with loss of heterozygosity - uniparental isodisomy (when the offspring inherits
both pairs of chromosomes from one parent), degree of relatedness of parents
SOLUTIONS GRADED A+ TIP
✔✔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
-mutant primer won't bind to the normal strand but wild type will always show up on the
mutant DNA
✔✔reverse transcription PCR - ✔✔-RNA cannot be used directly for PCR
-cDNA synthesis required to perform traditional PCR
-one step: reverse transcription (RNA to DNA) and PCR in one tube
-two step: cDNA synthesis first and then used as a target for separate PCR reaction
-allows detection of RNA based alterations such as exon skipping and RNA fusions
✔✔exon skipping from reverse transcription PCR - ✔✔-PCR primers designed across
the skipped exon show a size difference in comparison to end point PCR - missing a
section so they are smaller
✔✔RNA fusions from reverse transcription PCR - ✔✔-PCR primers designed to the two
different exons will normally not amplify but if the fusion product is present then the PCR
will amplify the abnormal mRNA
✔✔cytogenetics - ✔✔study of the strutter and function of chromosomes and their role in
human disease
-resolution of 5-10 mb
-highly subjective (different people see different things)
, -requires cell culture - cannot just be from tissues
-diagnostic yield is low
-good to test for aneuplodies but not other structural variants
✔✔constitutional cytogenetics - ✔✔diagnosis of heritable (germline) genetic
abnormalities in the prenatal, paediatric and adult settings
✔✔cancer cytogenetics - ✔✔detection of acquired (somatic) genetic abnormalities for
the diagnosis, prognosis, therapy, and/or monitoring for many types of cancer
✔✔variation in chromosome number and structure - ✔✔-aneuploidy and policy changes
- from errors occurring during meiotic or mitotic segregation
-translocation, inversions, insertions, deletions, duplications
✔✔balanced structural chromosome changes - ✔✔-no loss/gain of genetic material
-translocations
-inversions
-insertions
✔✔unbalanced structural chromosome changes - ✔✔-loss/gain of genetic material
-deletions
-duplications
✔✔paracentric vs pericentric inversion - ✔✔-para involves the centromere and peri
doesn't
-if the region inverted loops when the centromere is involved and there is recombination
then there will be loss or gain in the offspring
✔✔isodicentric chromosome - ✔✔-mirror image duplication involving the centromere
✔✔isochromosome - ✔✔-mirror image duplication not involving the centromere
✔✔ring chromosomes - ✔✔-when the chromosome is fractured and it tries to fix itself
-chromosome breaks in two places and the ends fuse together
✔✔chromosomal SNP microarray detection - ✔✔-genomic imbalances
-duplications and deletions (copy number loss)
-whole chromosomal aneuploidies
-triploidy (69 chromosomes)
-microdeletions and microduplications - CNV's and unbalanced rearrangements - loss of
heterozygosity - more related = more haplotypes the same
-mosaic imbalances >10% - when different cells in the body have different genotypes
-regions with loss of heterozygosity - uniparental isodisomy (when the offspring inherits
both pairs of chromosomes from one parent), degree of relatedness of parents
