Applications of PCR and NAH

Question 1

advantage of pcr

Answer 1

can be used for heterogenous samples

Question 2

reverse transcription pcr

Answer 2

Starting with mRNA
mRNA has a poly-A tail (a stretch of adenine [A] bases) at its 3′ end.

🧲 2. Anneal Primer
A primer made of oligo(dT) (e.g., dTTTTTTTTTTTTTTT or dT₁₅) is used.

It complements and binds to the poly-A tail of the mRNA.

This gives reverse transcriptase a starting point.

🔁 3. Reverse Transcription
Reverse transcriptase synthesizes complementary DNA (cDNA) using the mRNA as a template.

It extends from the 3′ end of the primer, creating a DNA strand complementary to the mRNA.

🧪 4. Alkali Treatment
The mRNA strand is degraded by alkali treatment, leaving only the cDNA.

Question 3

real time pcr purpose

Answer 3

semi-quantitative determination of the amount of a particular DNA/RNA species of interest present within a heterogeneous sample

Question 4

taq DNA polymerase

Answer 4

5’ to 3’ exonuclease activity

Question 5

real time pcr probe

Answer 5

attached with reporter (fluorophore) and quencher

During the annealing step of PCR, the probe binds to the target DNA between the primers.

During the extension phase, Taq polymerase encounters the probe and its 5’ to 3’ exonuclease activity cleaves it.
This separates the fluorophore from the quencher.
Once cleaved, the fluorophore emits a signal, which is detected.

The fluorescence intensity correlates with the amount of DNA amplified.

Question 6

digital pcr

Answer 6

enables an absolute quantification of DNA/RNA

Design microreactors in excess of dna in sample so only a single dna molecule will enter each reactor
Count number of positive pcr reactions for absolute quantification

Question 7

nucleic acid hybridisation

Answer 7

use of synthesized oligonucleotide probes fixed to a solid support to detect the presence of a DNA/RNA species of interest

Question 8

in situ hybridisation

Answer 8

detect the presence/localisation of DNA/RNA in cells/tissues following fixation to a solid support

Probes are usually fluorescently labelled (i.e. FISH)

Widely used to visualise the patterns of mRNA transcript localisation in developing embryos, tissue sections, and cell culture

Question 9

microarrays

Answer 9

genome/transcriptome wide analyses
design probes complementary to all protein coding genes in human genome.

silicon chip