Genetic Technology

Question 1

CMA

Answer 1

• chromosome microarray
• detects CNVs (missing or extra DNA/ “microduplications/microdeletions” 500 bp to 30 kb)
• Usually either aCGH and/or a microarray
• Can’t detect balanced rearrangements, SNVs, small indels

Question 2

aCGH

Answer 2

• array comparative genomic hybridization
• detects CNVs/indels as small as 100bp
• cannot determine locus or orientation of duplications
• can’t detect point mutations or balanced translocations

Question 3

WES

Answer 3

• whole exome sequencing genes and their introns
• can rarely detect balanced translocations
• unable to determine the phase of sequence variants (e.g., when two likely causative mutations for recessive disorders are found, cannot be certain that the mutations are on different alleles)

Question 4

WGS

Answer 4

whole genome seq

Question 5

Sanger seq

Answer 5

• sequences predetermined regions with primers
• can detect most SNVs within the sequenced region
• can detect insertions up to 300bp
• Can’t detect CNVs
• doesn’t sequence regulatory regions outside of genes
• Slippage can be an issue with runs of mononucleotide repeats

Question 6

SNP chip

Answer 6

• Often only used in DTC testing (e.g., for ancestry estimates)
• Usually looks at only 1% of genome
• Not “sequencing”

Question 7

MLPA

Answer 7

• Multiplex Ligation-Dependent Probe Amplification Assay
• Del/dup
• Detects small indels within a single exon of a given gene or within an entire gene
• The peak heights of the amplification products of the target DNA sequence is compared to the peak heights in reference samples
  
  • A del/dup is inferred from the relative decrease or increase in peak height
• Can’t detect point mutations

Question 8

karyotype

Answer 8

• can only detect large structural chromosomal rearrangements

Question 9

FISH

Answer 9

• Flourescence in-situ hybridization
• detects ploidy and larger CNVs
• Can’t detect locus of CNVs
• Can’t detect balanced rearrangements

Question 10

methylation analysis

Answer 10

e.g., for PWS/Angelman, etc.

Question 11

mtDNA seq

Answer 11

sequences mtDNA

Question 12

ms-MLPA

Answer 12

• Methylation-specific MLPA (MS-MLPA)
• Used to detect both the copy number and methylation status of DNA sequences in a single multiplex PCR-based reaction
• Target DNA sequences recognized by the MS-MLPA probes contain restriction sites for enzymes such a HhaI or HpaII that are sensitive to cytosine methylation of one CpG site in their recognition sequence
  
  • When target DNA is digested with these enzymes, the probe will amplify if theCpG site is methylated
  • The level of methylation is determined by the ratio of the relative peak area for each target probe from digested vs undigested DNA sample.
• Used for imprinting disorders like Prader-Willi syndrome /Angelman syndrome, Beckwith-Widemann syndrome, Russell-Silver syndrome, Lynch syndrome and Albright hereditary osteodystrophy.
• MS-MLPA has several advantages over other assays such as MS-PCR based on bisulphite sequencing, southern blotting, and methylation analysis including PCR following restriction digestion with methylation sensitive enzyme.
• Can’t detect point mutations

Question 13

repeat-primed PCR

Answer 13

uses specific primers amplify full-length PCR products from each allele, for disorders associated with presence or absence of a nucleotide repeat expansion (e.g., Fragile X, HD, etc.)

Question 14

Methylation specific PCR

Answer 14

pairs methylation sensitive DNA digestion with repeat-specific PCR amplification of the a repeat region to determine the methylation status of full mutation alleles