Lecture 16: PCR and NGS

Question 1

types of PCR

Answer 1

• PCR = polymerase chain reaction
• RT-PCR = reverse transcription PCR
• RT-qPCR = Real time PCR
• dPCR = digital PCR

Question 2

PCR use and issues solved

Answer 2

• simple and widely used to amplify and detect specific DNA sequences
• issues solved:
  o amplification – small sampling
  o specificity – 3.4 billion base pairs

Question 3

PCR reaction components:

Answer 3

• reaction buffer
• target DNA/template
• primers/oligos (predesigned, specific, forward and reverse)
• deoxynucleotides
• DNA polymerase

Question 4

Reverse transcription PCR:

Answer 4

• Use of RNA as a template
• DNA: stores info (chromosomes), inheritance, stable
• RNA: temporarily expresses info, profile constantly changes, degrades easily
• 2 step process:
  o RNA converstion to complementary DNA
  o Common PCR
• cDNA conversion:
  o reverse transcriptase
  o 3 enzymatic activities
• Used to measure gene expression via messenger RNA

Question 5

PCR/RT-PCR:

Answer 5

• Detects presence/absence
• Semi-quantitative at best
• Time consuming, low sensitivity and resolution, non-automated, short dynamic range, size-based discrimination only, qualitative results, not expressed as numbers.

Question 6

Real-time quantitative PCR:

Answer 6

• Measure quantity of DNA present at each cycle during PCR
• Quantification cycles (Cq) identify curve positions based on threshold crossing and starting quantity DNA.
• Each cycle, DNA amplification is detected using fluorescence
• Detection chemistries form basis of real time PCR flexibility and its multitude of applications

Question 7

• Detection of fluorescence

Answer 7

o Monitor fluorescence emitted during reaction as an indicator of amplicon at each cycle in real time

Question 8

• PCR system consists of 3 main components:

Answer 8

o Thermal cycler
o Optical module – scan plate using and records fluorescence
o Computer – translate fluorescence into meaningful data

Question 9

• Real time and normal PCR known as …

Answer 9

end point PCR

Question 10

PCR adv

Answer 10

rapid, no post-PCR processing, high throughput, increased sensitivity and dynamic range, high res, real time monitoring

Question 11

PCR prep

Answer 11

1. RNA extraction
2. cDNA conversion
3. preparation of samples (primer optimization)
4. plate set up
5. data analysis

Question 12

data analysis

Answer 12

o relative standard curve method
o comparative Ct method (fold-change)

Question 13

digital PCR (dPCR):

Answer 13

• 3rd gen PCR
• Based on sample partitioning
  o Droplet-based (Bio-rad)
  o Micro-well chambers

Question 14

digital PCR (dPCR): adv

Answer 14

high sensitivity and precision, greater tolerance to inhibitors, high reproducibility, increased number of clinical applications

Question 15

digital PCR (dPCR): disadv

Answer 15

high cost

Question 16

digital PCR (dPCR): applications

Answer 16

liquid biopsy analysis, viral load detection, copy number variation, rare mutations, gene expression analysis

Question 17

DNA sequencing:

Answer 17

• Determining the order of the four chemical building blocks/bases that make up the DNA molecule

Question 18

DNA sequencing: applications

Answer 18

o Discover genetic info carried in particular DNA segment and highlight changes that may cause disease – mutations

Question 19

1st generation NGS 1987

Answer 19

o High accuracy 99.99%
o High cost
o Low throughput
- Automation of sanger method
- Capillary electrophoresis
o Electropherogram results
o Computer data acquisition and analysis
- Fluorescent dyes, not radioactive
- Human genome project
- Key NGS feature is parallelisation of many reaction, achieved through sutomation and miniaturization
- High-throughput sequencing

Question 20

2nd gen NGS

Answer 20

• Pyrosequencing 1996
• Detection of luminescence from the release of pyrophosphate on nucleotide incorporation into the complementary strand
• 454 life sciences (2005, Roche)
  o Emulsion PCR
   DNA attached to beads
  o High-throughput
  o Quicker and cheaper
• Solexa 2007 illumina
  o First commercially available massive sequencing tech
  o Illumina dye sequencing (sequencing by synthesis)
  o 70-80% DNA sequencing market globally
• Adapters:
  o DNA insert sequence
  o Flow cell binding regions
  o I5/i7 indexes (sample identifier)
  o Sequencing primer binding site
• Workflow
  o Sample prep (DNA extraction)
  o Library preparation (fragmentation, addition of adapters)
  o Sequencing:
   Library loaded on flow cell
   Fragments hybridise
   Bridge amplification
   Incorporation of bases
   Sequencing cycles
   Image and record
  o Data analysis
   FASTQ file data output
   Alignment to reference genome
   Bioinformatics software

Question 21

NGS applications:

Answer 21

WGS
WES
RNA sequencing

Question 22

• Whole genome sequencing (WGS)

Answer 22

o Identify inherited disorders
o Characterise mutations that drive cancer progression
o Track disease outbreaks

Question 23

• Whole exome sequencing (WES)

Answer 23

o Sequence protein coding regions of the genome (2%)
o Contains 85% of known disease related variants
o Cost effective alternative to WGS

Question 24

• RNA-sequencing:

Answer 24

o Measurement of gene expression
o Identification of transcript isoforms and gene fusions

Question 25

• ChIP-sequencing

Answer 25

o Chromatin immunoprecipitation (ChIP)
o DNA-protein interactions
o Transcription factors and other proteins
o Histone marks

Question 26

• Single cell sequencing

Answer 26

o WGS, WES, RNA-Seq, ChIP-Seq