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DNA Techniques (Part 2)

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Date @02/11/2020 8:00 AM-10:00 AM

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Module BB MMB3001

What Is A Cycle Threshold (Ct) Value In qPCR?

In this article, I will explain what is meant by a Ct value in
quantification (real-time) PCR (qPCR). I will also discuss the
difference between Ct and Cq values. A video tutorial explaining
https://toptipbio.com/ct-value-qpcr/

Overview
End-point PCR

For DNA

One reading — allows us to follow the progress of the reaction

Semi-quantitative

Detect by ethidium bromide and seen under flourescent lamp

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 RT-PCR

For RNA

One reading per cycle

Quantitative

Flourescent probes that measure flourescent intensity

qPCR curve: The plateau phase indicates that there is no more product

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 The Ct number indicates at which cycle number the exponential phase picks up.

Add housekeeping gene for reference (e.g. Actin, tubulin, GAPDH)

Delta delta CT = Difference between treated and untreated Target Gene CT -

Difference treated and untreated Housekeeping Gene CT

High-resolution melt (HRM): PCR machine goes up from 55 to 90 deg C, at 1

deg per min. When it reaches the melting temperature of the product,
fluorescence disappears.

RT-PCR Primer Design

Target sequence = 150-300 bp

Primer size = 18-21 bp

GC content = 40-60%

Last 2-5 bases are C or G to bind strongly to sequence of interest

Span exon-exon or intron-exon junctions that are always epressed

together

Allows one to realise if sample has been contaminated by micro-organsims

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 Bacteria have no introns

If the sample readout is much larger than expected, there is probably

contamination

cDNA Synthesis
1. Extract RNA

2. Generate cDNA using Reverse Transcriptase and Random primers (hexamers)

3. Process (one cycle)

a. 10 mins at 70 deg C (remove and secondary structures fromed by RNA)

b. 15 mins at 42 deg C (binding of primer)

c. 5 mins at 95 deg C (denature reverse transcriptase enzyme and allow

separation of DNA and RNA strands from cDNA)

💡 Not all RNAs are converted at the same efficiency!

Digital PCR
A type of qPCR

Uses the same reaction mix

Instead of one tube, it uses multiple wells (100s)

Proportion of positive : negative wells

No reference standards and no endogenous controls (no need to generate

graphs for the absolute or relative quantification of PCR)

Sequencing
1. Sanger sequencing

2. High-Throughput Sequencing (a.k.a. Next Generation Sequencing)

3. Bisulphite Sequencing - methylation analysis

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 Sanger Sequencing
Each nucleotide has a coloured probe

A laser can detect the probe and converts it into the letter which symbolises a
nucleotide

High-Throughput Sequencing
Short reads sand bioinformatics to combine all the piece

Need multiple reads for a particular sequence to estimate accuracy

Can sequence many genes at once

Output is much greater than Sanger sequencing relative to the cost

There are three steps:

1. Library preparation

2. Library amplifiction

3. Sequencing

By different technologies (e.g. Illumina)

Bisulphite sequencing
The aim is to compare cytosine vs methyl-cytosine

Take RNA sample and divide it into two halves

Treat one half with bisulphite to convert cytosine to uracil (N.B. Methyl-cytosine
CANNOT be converted into uracil)

When sequecning, the converted cytosines read as Thiamines

Compare to original sequence: All remaining cytosines were protected by

methylation, while all the Cs that are now reading as Ts were unmethylated

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