Molecular diagnostics

What methods are associated with molecular diagnostics?

Polymerase chain reaction

Whole genome sequencing

Microarrays

What are the applications of molecular diagnostics?

Infectious disease

Genetics (hereditary diseases)

Is DNA or RNA a better source material and why?

DNA, because the bases are more protected

Where do we get our source material?

Blood, swab (genital,mouth…), faecal test

What are the principles associated with PCR?

Nucleic acids can be detected by staining and visualisation through gel electrophoresis.

The only way to know if a particular sequence of DNA is present is to selectively amplify it.

What do you copy in PCR and why?

The DNA fragment that you’re interested in, because finding one gene in a genome is really hard. It’s like trying to find a ‘needle in a haystack’.

What are some of the properties of DNA?

DNA double helix

Hydrogen binds between base pairs

Adenine and thymine

Guanine and cytosine

What is a primer?

A complimentary sequence to what you want to copy, that we design.

What happens during step one of PCR (denaturation)?

• High temperature breaks hydrogen bonds holding base

pairs together

• ‘Melts’ double-stranded DNA revealing bases in specific

order

What happens during step two of PCR (annealing)?

• At cooler temperatures, complementary bases can bind

• Oligonucleotide primers ‘match’ small regions of the target

area (gene of interest)

• They bind to the matching areas (anneal)

Primers must be designed so that one matches the sense

strand and the other matches the antisense strand

What happens during step three of PCR (extension and elongation)?

• Temperature raised to approximately 74°C

• Synthesis of new complementary DNA strand from 3’ end

of primer

Only regions where primers bound will be amplified/copied. So

it’s really important that they only match the region we’re

interested in

What happens during step four of PCR (Cycling)?

• In one cycle (denaturation; annealing; extension) we have

gone from one copy to two

• Cycle is repeated multiple times

and product number increases

exponentially

• Average PCR run is 40 cycles

How do we analyse the results of PCR?

The amount and size of the PCR product can be

visualised using staining and gel electrophoresis.

This visually confirms if our pathogen / gene of

interest / strain is present.

The brighter it is the more DNA that is present and therefore the more that was in the patient originally.

Known as semi-quantitative PCR

How doe qPCR differ to semi-quantitative PCR?

You take an image/measurement after every cycle rather than just at the end.

What is the difference between SYBR Green I and Taqman?

SYBR Green I- binds to all double stranded DNA, more cycles causes more DNA and therefore more fluorescence

Taqman- Only fluoresces if DNA we’re interested in is present

How can qPCR be quantitative?

Fluorescence can be monitored using a fluorometer

What is the Cq value?

The amount of fluorescence you need to be sure specific DNA is there.

What happens during reverse transcriptase PCR?

• Uses reverse-transcriptase enzyme to produce

double stranded DNA from RNA

• This provides template for normal PCR

• Can also be incorporated into qPCR = RT-qPCR

What is a microarray?

DNA is extracted and broken into pieces

Microarray contains thousands of DNA probes (specific

known sequences)

If DNA sample contains that sequence (gene) it will bind to

probe and fluoresce

Fluorescence is read and translated into genotype data

How should molecular diagnostics be used?

• A ‘PCR positive’ result by itself, without analysing the

complex scenario, can be unreliable, even misleading

• It is important to obtain a complete medical

understanding of the disease scenario

• Simultaneous application of molecular diagnostic

methods and classical approaches, is often still required

for a fully reliable diagnosis…………for now!