Sequencing and PCR

Question 1

What is DNA hybridisation?

Answer 1

Pulling apart 2 strands of DNA then label particular sequence and put back together to find complementary sequence.

Question 2

What is agarose?

Answer 2

Seaweed carbohydrates heated in buffer to dissolve.

Forms a gel through which a current can flow.

Polymerised agarose is porous and allows for separation of DNA by charge and size.

Question 3

How is agarose gel electrophoresis conducted?

Answer 3

Wells are made in the gel where DNA, tracking dye, and glycerol are added near the negatively charged cathode. -ve DNA migrates to positive electrode.

Question 4

What does the rate at which DNA migrates depend on?

Answer 4

Strength of electric field

Buffer

% of agarose in gel

Size of the DNA (smaller moves faster than large)

Question 5

How is DNA migration on electrophoresis related to size?

Answer 5

Linear DNA migration is inversely proportional to log10 of molecular size

Question 6

What kinds of dye are used for gel electrophoresis?

Answer 6

Dyes that bind DNA and fluoresce under UV light to allow visualization such as ethidium bromide and GelRed.

Question 7

How can we determine the approximate number of bases in a DNA sample?

Answer 7

Using standards and then determine size of sample relative to the standards

Question 8

What is the substrate for DNA polemerase?

Answer 8

Deoxynucleoside triphosphate

Question 9

What is required for DNA polymer formation (DNA replication)?

Answer 9

Free 3’-OH

DNA polymerase

dNTP

Template strand

Short double stranded NA

MG2+

Question 10

What is azidothymidine?

Answer 10

A nucleoside like molecule that doesn’t have an OH (Na+ instead) on 3’ end preventing elongation of NA strand.

Question 11

What is dideoxyribonucleoside triphosphate?

Answer 11

Molecule with a H instead of OH at 3’ end ending the elongation of nucleotide sequence. This is used for Sanger sequencing

Question 12

How are nucleotides labelled in modern times?

Answer 12

Using radiolabels or a fluorescent dye

Question 13

What is PCR?

Answer 13

In vitro amplification of a region of DNA whose sequence is known or which lies between 2 regions of known sequence

Question 14

How is DNA polymerase in PCR stable at higher temperature?

Answer 14

Due to Taq polymerase created by thermostable bacteria in hot springs of yellowstone national park.

Question 15

What is required for PCR?

Answer 15

DNA template to copy

Primers to provide 3’OH

Enzyme (Taq DNA polymerase)

dNTPs

Mg2+

Buffer

Thermocycler (cycles between high temperature and low temperature)

Question 16

Why is a thermocycler necessary for PCR?

Answer 16

To allow the DNA to separate and then it is cooled to allow ligase to bind some primers to the DNA strand and then the strand can replicate at hot temperature.

Question 17

What are the types of primers needed for PCR?

Answer 17

2 sets: Forward and reverse. One binds to each template strand.

Question 18

Why are primers not made up of inverted repeats ever?

Answer 18

To prevent formation of hairpins.

Question 19

What bases make up primers for the most part?

Answer 19

GC is preferred for best annealing. (40 - 60% of bases)

Question 20

What part of the DNA are primers complementary to?

Answer 20

3’ ends of target DNA. They are synthetically produced.

Question 21

Taq DNA pol has 5’ - 3’ activity only and does not proofread. Is that an issue for PCR?

Answer 21

No, PCR is a qualitative assessment of a gene so if a few are mutated that isn’t an issue.

Question 22

What are the 3 stages of PCR?

Answer 22

Denaturation of DNA +/- 95 degrees (templates)

Primer hybridisation/annealing +/- 50 degrees

DNA synthesis (extension) +/- 72 degrees

Question 23

At what rate are sequences amplified in PCR?

Answer 23

Exponentially.

Question 24

How are PCR amplification products identified?

Answer 24

Via gel electrophoresis

Sequencing of amplified fragment

Southern blot

Question 25

What is PCR used for?

Answer 25

Diagnostics

Evolution studies

Detection of drug resistance genes

Forensics (DNA fingerprinting)