Topic 8

Question 1

What is polymerase chain reaction (PCR)?

Answer 1

Uses DNA template, primers, dNTPs, DNA polymerase (Taq or Pfu)
Cycles of temperature: denaturation (95C), annealing of primers (50-65C) and extension (polymerase) (72C)
Billion fold can be readily achieved in 2.5 hours
If we know the flanking regions of a gene: a more direct method for gene isolation and amplification

Steps: in a tube, add DNA template, primers, dNTPs, and DNA polymerase, then heat at 96-98 to denature, then cool down to 50-65C for primers to anneal, then heat at 72 to extend new strands, repeat previous steps, after many cycles many copies made

Slides 4-6

Question 2

What are the 2 methods for DNA sequencing?

Answer 2

1. Dideoxy sequencing (Sanger sequencing)

2. Next - generation (whole genome sequencing)

Question 3

What is Sanger sequencing?

Answer 3

Chain termination method
Primer binding sequences + DNA polymerases + dNTPs + ddNTP (stops synthesis, they are coloured)

Still widely used
Small scale projects

Slides 8-11

Question 4

What is “next-generation” whole genome sequencing?

Answer 4

First step: DNA fragmentation and denaturation
PCR is important tool again
Sequencing by synthesis
Reactions take place on beads in tiny walls

Pyrosequencing:
Based on detecting synthesis reactions
When a nucleotide is added, release of pyrophosphate
2 enzymes are present: sulfurylase and luciferase
Reaction: visible light
Different fluorescence light every time a nucleotide is inserted

Slides 12-15

Question 5

What is bioinformatics?

Answer 5

Study of the information content of genomes
With millions of reads, you can assemble CONTIGS
CONTIG is a set of overlapping DNA segments that together represent a consensus region of DNA
Using CONTIGS allow their assembly into scaffolds

Slide 16-19

Question 6

Study the structure of the genome slide 21

Answer 6

Ok

Question 7

How can DNA copies of mRNA be synthesized?

Answer 7

Eukaryotic genes often contain introns (human insulin contains 2)
Bacteria do not have the ability to splice out introns 
We are interested only in the coding sequence, we can use insulin mRNA as a starting material for PCR
complementary DNA (cDNA) is a DNA version of an mRNA molecule (reverse transcriptase)
Purifying mRNA from a tissue that produces large amount of desired protein 
mRNA is added to a tube with reverse transcriptase, the four dNTPs, and a short primer of polymerized dTTP residues 

Slide 22-23

Question 8

What is the transcriptome?

Answer 8

Eukaryotic genes contain introns
Introns are removed in mRNA
Remember the poly A tail? Oligo-day binds here
Reverse transcriptase synthesize the new DNA strand
Then, RNA is removed (chemically)
A DNA polymerase copies the cDNA strand (as in the PCR process)
cDNA library - next generation sequencing

Slide 24-26