1-24 prework Genetic Testing

Question 1

Human Genome Project

Answer 1

3 billion base pairs of “reference sequence”, therefore dipoloid genome 6 billion

techniques: mapping mRNA and expressed sequences to genome, computational tools to predict coding sequence, comparative genomics to other known species genomes, ENCODE project to ID regulatory sequences within the genome

20,000 genes mapped to human genome, less than people anticipated in 2001- only 1.5% is highly conserved coding, 3% highly conserved other, 45% transponson based repeats, 6% heterochromatin, 45% other non-conserved

lots of “junk” “genomic dark matter” DNA, poorly understand this dna

Question 2

Mitochondrial genome

Answer 2

93% highly conserved coding, 5% highly conserved other, less non-conserved

Question 3

clinical utility

Answer 3

evidence of improved measureable clinical outcome, requires “clinical validity” (relationship between the test result and the disease”,

clinical validity requires “analytical validity”, which is test accuracy

Question 4

Methods to detect known genetic variations

Answer 4

1. primer/probe design (requires prior knowledge of sequence)
2. amplification (target or signal)
3. detection (size or sequence variation)

Question 5

polymerase chain reaction

Answer 5

first amplification method

realtime pcr- quantitative PCR, can make standard curve and quantify amount of starting material in unknown samples

Question 6

methods to detect novel sequence variation

Answer 6

1. sanger dideoxy chain terminating: 10% of nucleotides dideoxy so sequence gets terminated and you run on gel or flourophore- you cannot quantify amount of mutant allele presence and you can only detect down to 10% of mutant allele is present (mosaicism), you can detect novel variants in coding region, you cannot detect whole exon dels/dups
2. Pyrosequencing: similar to sanger but via release of light is used to track, this also gives you quantitative data, useful for short sequences
3. Massively parallel sequence (NextGeneration, NGS): used pyrosequencing in a massively parallel region, 1. create library 2. ligate adaptors 3. capture and amplify 4. sequence 5. trim and align 6. analyze; can be quantitative, has low limit of detection, and removes cis-trans ambiguity, there are some errors so you want enough reads of strands to remove those erros

Question 7

Sequencing limitations

Answer 7

many known mutations are non-conding, non-splice site so we won’t detect them by traditional exon sequencing

primers are designed to amplify segmentsi ncluding coding sequence and slice sites only, therefore large deletions will not be detected

Question 8

Massively parallel sequence (NextGeneration, NGS):

Answer 8

used pyrosequencing in a massively parallel region, 1. create library 2. ligate adaptors 3. capture and amplify 4. sequence 5. trim and align 6. analyze; can be quantitative, has low limit of detection, and removes cis-trans ambiguity, there are some errors so you want enough reads of strands to remove those errors

not at clinical grade detection of single nucleotide changes but getting better

limitations: certain muts are not detectable, certain regions are not covered, analytical validity does not equal clinical validity

Question 9

Transition
Transversion
missense
nonsense
silent

Answer 9

purine for purine (vice versa)
purine for pyrimidine (vice versa)
change in single AA
codes for stop codon (stop gain)
codes for same aa (synonymous): this could still give a splice site change