Amplification and Detection of DNA Sequences

What you should know• Polymerase Chain Reaction (PCR) is the amplification of

DNA using complementary primers for specific target sequences.

• DNA heated to separate strands then cooled for primer binding.

• Heat-tolerant DNA polymerase then replicates the region of DNA.

• Repeated cycles of heating and cooling amplify this region of DNA.

• Arrays of DNA probes are used to detect the presence of specific sequences in samples of DNA.

• The probes are short single stranded fragments of DNA that are complementary to a specific sequence.

• Fluorescent labelling allows detection. • Applications of DNA profiling allow the identification of

individuals through comparison of regions of the genome with highly variable numbers of repetitive sequences of DNA.

Polymerase Chain Reaction (PCR)

• PCR has made it possible to analyze and characterize DNA fragments found in minute quantitiesminute quantities in places like a drop of drop of bloodblood at a crime scenecrime scene or a cellcell from an extinct dinosaur.extinct dinosaur.

The polymerase chain reaction(PCR)

The problem!

Maisy the research meerkat is pregnant, but

who’s the daddy?

How can we find out?

A real problem for a real scientist

Johanna Nielsen, a PhD student at the University of Edinburgh and the Institute of Zoology at the University

of Cambridge

What did she do?

She knew that DNA is the genetic material of living things

Therefore the DNA sequence of the foetus would have greater similarity with the father than with a male meerkat

who was not the father

She had two candidate fathers and decided to compare the DNA from the foetus with that of the two possible

fathers

She decided to use DNA profiling (fingerprinting) techniques, which involve the use of the polymerase

chain reaction, or PCR for short

Johanna knows that this section of DNA can be of different sizes between meerkats. She wants to compare the DNA at this site between the three samples.

The DNA profiling technique: compares the size of DNA at a specific site between individuals

DNA

Firstly, she needed to extract the DNA from each potential father and the

foetus.

What would she need to do to get a DNA sample?

By using PCR, Johanna can amplify this section of DNA many times.

PCR is now fully automated, so Johanna just needs to put the DNA samples into separate tubes in the PCR machine, which is called a thermo cycler, along with a PCR ‘master mix’, which includes all of the ingredients the PCR reaction needs.

•PCR uses the same mechanism for copying the DNA as our cells use for DNA replication. •In pairs, remind yourselves of what happens during DNA replication.

•What is the main enzyme involved?

PCR uses the enzyme DNA polymerase to replicate or ‘amplify’ the DNA strand.

Step 1: DNA denatured

Primers are used to start the replication process of just the

section needed.

Primers are short single strands of DNA that bond or ‘anneal’ to the sections of DNA on either side of the section of DNA that is wanted. The temperature is

reduced for this step

The DNA is heated to 94°C to denature the

double helix so amplification can take

place.

Primers anneal

Step 2: Primers anneal

The polymerase chain reaction

DNA polymerase

+Two strands of DNA are produced. Each piece of DNA consists of a template and a new strand, therefore the process is semi-conservative.

Annealed primers

Step 2: DNA

is copied (Temperature is increased to 72°C)

And then start the three steps all over again! ...but this time the amplified DNA is also used as a

template...

To give four...

This cycle is continued for 30–40 cycles.

If there were 35 cycles from one template strand, how many copies would you have after

the final cycle? (You might need your calculator!)

A lot!

With just one template you would get

34,359,738,368 copies after 35 cycles.

You will usually start off with more than one DNA template from a

DNA extraction. Why?

Each cell has a copy of the DNA and an extraction will be from

many cells.

A recap...

30-40 cycles

Put the following steps involved in PCR into the correct order.

• DNA replication occurs using DNA polymerase.• Primers introduced and temperature reduced to allow bonding.• Temperature increased again to separate strands and the

cycle begins again.• Temperature increased to 95°C to separate strands.• Double-stranded DNA produced, consisting of a template and

a new strand of DNA.

Draw an annotated diagram to accompany each stage.

Share your finished note with a partner and check the steps are in the correct

sequence

• Temperature increased to 95°C to separate strands.

• Primers introduced and temperature reduced to allow bonding.

• DNA replication occurs using DNA polymerase.• Double-stranded DNA produced, consisting of a

template and a new strand of DNA.• Temperature increased again to separate

strands and the cycle begins again.

Back to Maisy, who is still pregnant!

Maisy’s baby

Malcolm Martin

After PCR what will be in the sample tubes?

Millions of copies of the DNA section/fragment selected by Johanna.

Remind yourself why Johanna wanted to amplify this fragment.

Johanna needs to find out the size of the DNA fragments inside each sample.

She uses a technique called DNA gel electrophoresis, which separates DNA fragments based on their size.

In the past this was done in tanks, using agarose gel electrophoresis, but laboratories now use an automated machine that can process many samples quickly using capillary gel electrophoresis.

The results are then processed by a computer, producing data like that shown above. This example shows the results of two different PCR samples. One of the DNA fragments is 121 base pairs in length, the other is 159 base pairs.

If the PCR sample contains millions of DNA fragments, why would one sample only produce one data peak?

Yes, that’s right, the PCR reaction will amplify the same fragment many times. The technique, however, is not perfect and that is why sometimes you get the extra little peaks that you can see in the data above.

Here are Johanna’s data for three different DNA fragments from each of the meerkats

Malcolm

Martin

Who is the daddy?Martin!

To make copies of the DNA you need to add to the PCR mix:

DNA polymerasenucleotides of each base type.primer

PCR uses a special DNA polymerase – Taq polymerase

Why?+

Where is Taq from?+

What is one of the problems with using it?

Taq polymerase is stable at high temperatures and is therefore perfect for PCR where the first step of each cycle requires temperatures of 94°C in order to denature the DNA.

Taq comes from a thermophilic bacterium, Thermus aquaticus, which lives in hot springs or hydrothermal vents, hence its high temperature tolerance.

The main problem with using it in PCR is that it doesn't have a mechanism for proofreading the DNA. This can lead to errors that are not corrected. Remember that PCR produces millions of copies from just one template. If a copy contains an error, for example a change in one of the base pairs, then that error will be copied millions of times, which is especially problematic if it occurs in an early cycle.

PCR Summary

DNA probes

Once DNA has been amplified how is it searched?

What are DNA probes?

DNA probes are pieces of DNA used to detect the presence of specific sequences in DNA. (Do not get probes mixed up with the primers used in PCR.)

What characteristics will DNA probes need to have?

DNA probes

• Made of DNA

• Short

• Single stranded

• Complementary to the sequence being searched for

How do we find where the probes have bound?

Before the DNA probe is introduced to the DNA it is fluorescently labelled. This involves attaching a fluorescent dye to the probe. For example, ethidium bromide fluoresces orange when bound to DNA and exposed to UV light.

Medical and forensic applications

In groups discuss how DNA probes along with PCR can be used to:

•make a diagnosis of disease status or risk of disease onset

•identify if a particular individual left blood at a crime scene.

Making a diagnosis of disease status or risk of disease onset

•A cell sample from a patient can be screened for the presence or absence of a particular sequence, eg a mutation in a gene. •This information can be used to diagnose a condition or describe the likelihood of a condition developing. •For example, women with a family history of cancer can be tested for mutations in the BRCA 1 and BRCA 2 genes. •Mutations in these genes can increase the risk of developing breast or ovarian cancer.

Identifying if a particular individual left blood at a crime scene

•DNA profiling allows the identification of individuals through comparison of regions of the genome with highly variable numbers of repetitive sequences of DNA.•So if a tiny sample of blood is left at a crime scene, the DNA it contains can be amplified using PCR. •A probe can then be created, which binds to one of these highly variable sequences. •If a suspect is apprehended their DNA can be tested with the same probe to look for the sequence in question.

Do you know ?• Polymerase Chain Reaction (PCR) is the amplification of

DNA using complementary primers for specific target sequences.

• DNA heated to separate strands then cooled for primer binding.

• Heat-tolerant DNA polymerase then replicates the region of DNA.

• Repeated cycles of heating and cooling amplify this region of DNA.

• Arrays of DNA probes are used to detect the presence of specific sequences in samples of DNA.

• The probes are short single stranded fragments of DNA that are complementary to a specific sequence.

• Fluorescent labelling allows detection. • Applications of DNA profiling allow the identification of

individuals through comparison of regions of the genome with highly variable numbers of repetitive sequences of DNA.