Lecture 18

Question 1

Copying DNA in a tube

Answer 1

Ingredients: template, DNA polymerase, primers (forward and reverse), deoxyribonucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, dGTP) and buffer (pH and ionic strength and Mg2+)

Question 2

Copying DNA in a tube vs in cell

Answer 2

Simpler in tube than cell as they need enzymes, synthesise RNA primers and removes them, fill in nick, maintain pH, make dNTPs.

Question 2

PCR steps

Answer 2

1. Mixture is heater to separate DNA ~90º (replaces helicase). As DNA separates, abs. increases (increases % G and C, increase length, ionic strength = increase Tm)
2. Annealing occurs where the DNA is cooled to allow specific primers form H bond to 3’ OH. They are specifically made to amplify area you want. You also want them to face the right way.
3. Extension: Polymerase catalyses formation of phosphodiester bond, adding nucleotide to growing chain
4. Cycling: The process is repeated again with the newly synthesised strand forming the template for the next. In cell, repetition doesn’t occur.

Question 3

Designing primers

Answer 3

Appropriate Tm must be similar to the forward and reverse primers so they can both anneal to DNA and not itself or each other or each other. Can be determined by length and sequence (GC content, presence of complementary sequence). Increase Tm = harder to get sequence to anneal.

Question 4

Mg2+

Answer 4

Required for DNA polymerase function. Positive therefore, shields negative phosphates decrease repulsion between two strands meaning they will be more likely to stay together (promotes base pairings). Primers will be less specific as specificity comes from unfavourable ionic interactions which is reduced in presence of Mg2+.

Question 5

Taq polymerase

Answer 5

Heat stable. Able to work after heating to 95ºC with optimal temp. at 72ºC.

Question 6

Electrophoresis

Answer 6

Sugar-phosphate backbone is - charged. As it is continuous unit, constant charge:mass. Can separate DNA by size where smaller fragments travel further than larger ones.

Question 7

Staining

Answer 7

Dyes can help visualise DNA/RNA. Ethidium bromide intercalates with DNA and fluoresces under UV light. ‘Safer’ dyes replace it.

Question 8

Genotyping

Answer 8

Length of variable number tandem repeats will vary between individuals. Primers bind to region that’s common to everyone and moves in to amplify variable region which is different for people. Size of amplified DNA increases with no. repeats.

Question 9

Reverse transcriptase

Answer 9

RNA is hard to work with. To make it easier, you can copy it into DNA (cDNA) achieved by reverse transcriptase. Also used by retroviruses. It needs primers which can be chosen based off application.

Question 10

Contamination

Answer 10

Ribonucleases found on common surfaces. RNA contaminated with ribonucleases will degrade therefore convert to cDNA quickly or don’t.

Question 11

RT-PCR

Answer 11

Looks at how much mRNA is made of a target gene. RT = reverse transcriptase. Two stages of DNA with sequence-specific primers. COVID-19 isolate RNA to make cDNA to PCR gene of interest to analysis.

Question 12

Sanger sequencing

Answer 12

1. Primers bind close to target region.
2. DNA Polymerase, four dNTPs and fluorescent ddNTPs added.
3. DNA is amplified and tagged with ddNTP preventing further amplification.
4. DNA is denatured.
5. Gel electrophoresis and further analysis shows DNA sequence.

Limitations on length and need info about sequence for primer design. Useful for detecting mutations.