Lecture 7: PGx testing II Flashcards
Goal of testing
-testing known variants
-testing known and unknown alleles
Genotyping
-testing known variants
-DNA chip
Sequencing
-testing known AND unknown alleles
-sanger sequencing**
-high-throughput next-gen sequencing (whole exome sequencing)**
Fundamental technique for DNA amplification
PCR
DNA amplification (not starrred)
-DNA too big to look at whole thing
-target sequence
-amplify target
-allele discrimination (hom vs het)
Polymerase Chain Reaction (PCR)
-amplify specific region of genome making billions of copies detectable
-enzymatic reaction
-substrates
-product = DNA molecules that start and end w primers
PCR substrates
-DNA template
-dNTPs (dATP, dGTP, dCTP, dTTP)
-primers: 2 short seq specific to region of interest
-buffer pH and Mg2+
-taq DNA polymerase enzyme
PCR steps
-denaturation (inc temp)
-Annealing (dec temp to let primers base pair to DNA)
-Extension (polymerase extends primer to dorm nascent DNA strand)
-Exponential Amplification (repeat 30 cycles to make 2 billion copies)
PCR reaction
-chain-reaction
-from 2 copies to 2^n-1 copies (n= number of cycles)*
-taq polymerase thermal stable enzyme
Important PCR not
-PCR amplifies both DNA molecules of homologous chromosomes
-this is why you can tell a genotype
-PCR rxn products (amplicon) are mixture of double-strand DNA products generated from both chromosomes (primers equally bind each chromosome)
-need additional specific technique to distinguish each allele
DNA chip
-detect known SNPs
-high throughput
-medium cost
-larger-scale used for research (genome-wide based studies)
-mid-throughput for PGx testing
Chip-based PGx testing platform (not starred)
-amplichip CYP450 Array
-diagnostic device that covers CYP2D6 and CYP2C19 in single test
DNA (Sanger) Sequencing
-conventional
-low throughput
-targeted seq: one specific DNA fragment
-based on selective incorporation of chain-terminating ddNTPs by DNA polymerase during in vitro DNA replication
-developed by Sanger in 1977 (only person to win chem nobel prize twice (insulin))
Next generation sequencing
-high throughput
-parallel
-massive (seq multiple DNA frags at same time)
How to read Sanger seq
-read from bottom to top
-phoresis
-look at slide
Allele discrimination in Sanger Sequencing
-PCR amplificatin
-change color of waves idk (half blue + half red = heterozygous)
Sanger Sequencing
-can detect both known and unknown alleles (SNPs, indel, camll CNV)
-low throughput
-96 samples per overnight for one DNA frag
-higher cost per base pair and per SNP
-widely used in PGx testing
Next generation Sequencing steps
-seq by synthesis in parallel
-select DNA frags
-attach adapters to create seq library
-apply to flowcell
-clustern generation by solid phase PCR (bridge amplification) = synthesis in parallel
-seq by synthesis with reversible terminators
Next gen seq (NGS)
-high throughput (can simulataneously seq DNA of multiple individuals
-can target whole genome/exome vs targeted genes
-higher cost
-detect all known or unknown alleles
-detect almost all kinds of polymorphisms
-inc use in clinical PGx testing
Sequencing depth and coverage
-average # of reades that align to, or cover known reference bases
-NGS coverage level often determines whether variant discovery can be made with a certain degree of confidence at particular base positions
Recommended seq depth/coverage for detecting human mutations, SNPs, rearrangements
-10^x - 30^x
NGS shotgun requires seq every base in a smaple several times for two reasons
-need multiple observations per base to come to a reliable call
-reads not distributed evenly over entire genome bc reads will sample genome in random and independent manner
