Lec 6-2

Question 1

PCR reaction mixture inside a tube

Answer 1

DNA template (double stranded)

Primers (forward and reverse)

dNTPs

DNA polymerase (heat resistant)

Buffer (ions)

Question 2

Steps of PCR

Answer 2

Separating double stranded DNA with heat

Primers anneal to single strands of DNA by cooling

DNA synthesis by polymerase

Question 3

Steps of PCR

Separating double stranded DNA with heat

Answer 3

Heat to 100 degrees for a few minutes

Question 4

Steps of PCR

Primers anneal to single strands of DNA by cooling

Answer 4

Cool rapidly for less than a minute

Primers are added in high concentrations at 5’ end of each strand and able to bind to template before larger single stranded DNA comes back together

Both forward and reverse primer is used to flank a sequence of interest

Question 5

DNA synthesis by polymerase

Steps of PCR

Answer 5

Heat to 72 degrees, allowing for DNA synthesis to occur from the primers

Question 6

PCR thermocycler

Answer 6

Allows PCR steps to be repeated many times (25-35)

Question 7

Result of PCR

Answer 7

Robust amplification of DNA flanked by primers

Amplifies one specific sequence

Question 8

DNA sequencing and DNA libraries capabilities

Answer 8

Locate a gene/DNA sequence

Question 9

Every cell in our body have

Answer 9

The same genetic code

Question 10

On average how many genes in the genome are expressed

Answer 10

1/10th

Question 11

Creating cDNA (complementary DNA) libraries

Answer 11

Cell, tissue or organism is placed into a tube with mRNA

TEXTBOOK

Question 12

DNA sequencing gives us the ability to

Answer 12

Discover the precise DNA sequence of previously unknown identity

Question 13

Sanger sequencing background

Answer 13

Created by Frederick Sanger

Standardly used sequencing DNA fragments in laboratories

Also called Didoexy Sequencing

Question 14

Sanger sequencing similarity to PCR

Answer 14

DNA replication with DNA polymerase

DNA template

Primers

Question 15

Sanger sequencing differences to PCR

Answer 15

Only one primer

Relies on addition of special nucleotides, ddNTPs in addition to dNTPs used in PCR

ddNTPs are much lower than dNTPs

Either primer or ddNTPs are labeled with a radioactive isotope or fluorescent probe, the synthesized fragments are separated via electrophoresis and imaged to readout sequence

Requires large amount of DNA

Question 16

ddNTPs (dideoxyribonucleoside
triphosphates)

Answer 16

Lack the 3’ hydroxyl group of dNPTs which is critical for the addition of new nucleotides by DNA polymerase

Question 17

Sanger sequencing original version

Answer 17

Primer is radioactively labelled on the 5’ end

Many copies of DNA and primer are added to 4 tubes

Very expensive and laborious

Question 18

Sanger sequencing original version

Each tube receives

Answer 18

A lot of dNTPs

DNA polymerase

A small amount of the 4 ddNTPS (A C G T)

Question 19

Sanger sequencing

automated version

Answer 19

Primer is not labelled

Each ddNTP (A C G T) is labelled with a difference fluorescent probe

Everything goes into one tube and one well on gel

Carried out by automated machines using laser scanners, imagers and software to read out DNA sequence