PGx Testing Technology II

Question 1

PGx Testing Methods

Answer 1

Testing the KNOWN variants
- Genotyping: DNA Chip

Testing both KNOWN and UNKNOWN
- Sequencing: Sanger & High Throughput Next-Generation Sequencing

Question 2

Fundamental Technique for DNA amplification

Answer 2

PCR

Question 3

Polymerase Chain Reaction (PCR)

Answer 3

the most useful technique for DNA amplification: 50-1000 bp

Function: amplify a specific region from the genome to make billions of copies (2^35) –> detectable

Type of Reaction: enzymatic

Substrates:
- DNA template
- dNTP (dATP, dGTP, dCTP, dTTP)
- 2 primers (allow initiation of the reaction and specific for the region of interest)
- Buffer: pH & Mg2+
- Taq DNA polymerase

Products:
- DNA molecules

Question 4

Process of 3 Water Bath

Answer 4

Denaturation:
- temperature is increased to 98 to separate DNA strands

Annealing:
- temperature is decreased to 48-72 to allow primers to base pair to form DNA template

Extension:
- polymerase extends primers to form nascent DNA strand

Question 5

Important Point

Answer 5

PCR amplifies DNA from both DNA molecules of homologous chromosomes (2n) simultaneously

Question 6

DNA Chip

Answer 6

What? detect KNOWN SNP or target SNP

high throughput
- up to 5M SNPs can be genotyped simultaneously

low cost per SNP

genome-wide based studies used for research

Question 7

Types of DNA Sequencing

Answer 7

Conventional Sequencing (Sanger)
- low throughput
- targeted sequencing: one specific DNA fragment

Next Generation Sequencing
- high throughput
- parallele sequencing
- massive sequencing: multiple DNA fragments

Question 8

Who developed Sanger Sequencing

Answer 8

Frederick Sanger in 1977

won 2 nobel prizes

Question 9

What is Sanger Sequencing?

Answer 9

method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTP) by DNA polymerase

Question 10

Sanger Sequencing

Answer 10

What? detect both KNOWN and UNKNOWN (SNP, indel, CNV)

low throughput

higher cost per base pair compared to DNA Chip

higher cost per SNP compared to DNA Chip

Question 11

Next Generation Sequencing

Answer 11

What? detect all KNOWN and UNKNOWN

high throughput
- simultaneously sequence DNA of multiple individuals

can be used for whole genome/exome

higher total cost

very low cost per SNP

VERY FAST BUT CAN HAVE ERRORS

Question 12

Sequencing Depth/Coverage

Answer 12

determines whether a variant discovery can be made with a certain degree of confidence at particular positions

10x to 30x depth of coverage is recommended (how many times you run the reaction)

Question 13

Why does NGS require sequencing of every base several times?

Answer 13

1. multiple observations are needed per base to come to a reliable base call
2. reads are not evenly distributed evenly over an entire genome, simply because the reads will sample the genome in random & independent

Question 14

Germline vs Somatic

Answer 14

Germline
- sequence of germ cells that may be passed to child
- exists in somatic genome as well
- exists since the individual was born

Somatic
- sequence of nongermline cells that are NOT passed to child
- does not exist in germline genome
- acquired (cancer, sun induced, etc)

Question 15

Detection Methods of Somatic Mutations

Answer 15

Sanger: point mutations, small indels

NGS: all kinds

NO DNA CHIPS BECAUSE THEY ARE UNKNOWN

Question 16

HIPPA

Answer 16

Health Insurance Portability and Accountability Act
- opt in and opt out is an evolving concept
- GINA (genetic information non-discrimination act)