Recombinant DNA 3

Question 1

How to make RNA in vitro ?

Answer 1

1. A vector (DNA molecule) has a T7 promoter
2. RNA polymerase transcribes the DNA into RNA

Question 2

How to make an RNA vaccine against SARS-Cov2 ?

Answer 2

1. Insert gene encoding virus spike protein into suitable plasmid vector
2. Transcribe in vitro, with uridine replaced by pseuodouridine
3. Add 5’ cap and 3’ poly A tail to RNA
4. Remove vector DNA, purify spike protein mRNA
5. Package pure spike protein into lipid nanoparticles

Question 3

What are methods to detect specific DNA and RNA molecules ?

Answer 3

Molecules are identified using a probe that hybridises
The probe hybridises only to the complementary fragment
Blotting methods

Question 4

What are blotting methods ?

Answer 4

The DNA/RNA is separated by gel electrophoresis, then transferred to a membrane and hybridised with a labelled probe.
It can be used to determine the extent of gene expression.

Question 5

What is a southern blot ?

Answer 5

Separates DNA
Probe labelled DNA or RNA

Question 6

What is a northern blot ?

Answer 6

Separates RNA
Probe labelled DNA or RNA

Question 7

What is a western blot ?

Answer 7

Separates proteins
Probe is antibody

Question 8

What is a DNA profile ?

Answer 8

A set of genetic markers (usually short tandem repeats) that are highly variable between individuals. They are analysed and used to compare DNA samples.

Question 9

What are STRs and why are they used in DNA profiling ?

Answer 9

They are short sequences of DNA that are repeated in a row
They are used in DNA profiling because:
1. Highly variable between individuals
2. Stable inheritance

Question 10

Describe DNA profiling

Answer 10

1. STRs amplified by PCR
2. Analysed by capillary electrophoresis, and the number of of repeats is shown
3. DNA profile is compared to known DNA to find a match

Question 11

What is qPCR ?

Answer 11

Measures the amount of DNA formed
Uses real time PCR

Question 12

What is qRT-PCR ?

Answer 12

Quantitative reverse transcription PCR
Detects presence of RNA by converting it into cDNA

Question 13

How does quantitative real time PCR work ?

Answer 13

A fluorescent dye or probe is added to the DNA
Then during PCR the dye emits light during each amplification cycle, which is measured in real time

Question 14

What is the cycle threshold (Ct) ?

Answer 14

The number of cycles required to reach a defined threshold quantity of product is determined

Question 15

How is RT-PCR used to diagnose covid ?

Answer 15

It interprets the Ct values to determine the viral load present
Low Ct value = high viral load