1. Amplification and Detection of DNA Sequences

2. 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.

3. Polymerase Chain Reaction (PCR)
PCR has made it possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.

4. The polymerase chain reaction (PCR)

5. Maisy the research meerkat is pregnant, but who’s the daddy?
The problem!
Maisy the research meerkat is pregnant, but who’s the daddy?
How can we find out?

6. A real problem for a real scientist
What did she do?
Johanna Nielsen, a PhD student at the University of Edinburgh and the Institute of Zoology at the University of Cambridge

7. 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

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

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

10. 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.

11. 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?

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

13. The polymerase chain reaction
The DNA is heated to 94°C to denature the double helix so amplification can take place.
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
Primers anneal
Step 2: Primers anneal

14. + Step 2: DNA is copied (Temperature is increased to 72°C)
DNA polymerase
Step 2: DNA is copied (Temperature is increased to 72°C)
Annealed primers +
Two strands of DNA are produced. Each piece of DNA consists of a template and a new strand, therefore the process is semi-conservative.

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

16. 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!)

17. With just one template you would get
A lot!
With just one template you would get
34,359,738,368 copies after 35 cycles.

18. You will usually start off with more than one DNA template from a DNA extraction. Why?

19. Each cell has a copy of the DNA and an extraction will be from many cells.

20. A recap...
Just to recap
30-40 cycles 20

21. 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.

22. 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.

23. Back to Maisy, who is still pregnant!

24. After PCR what will be in the sample tubes?
Maisy’s baby
Malcolm
Martin
Millions of copies of the DNA section/fragment selected by Johanna.
Remind yourself why Johanna wanted to amplify this fragment.

25. In the past this was done in tanks, using agarose gel electrophoresis,
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.
This machine uses capillary gel electrophoresis which uses the same principles as gel electrophoresis carried out in electrophoresis tanks, apart from the tank is replaced by very thin capillaries which contain a polymer instead of agarose. The machine detects the fragments, using a laser, as they pass through the capillary. The fragments are labelled with fluorescent tags which are attached to the primers in the PCRreaction.
This machine is made by Applied Biosystems, whose website gives further information about their models. 25

26. 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.
This is real data. The graph shows two main peaks from two different samples. The x axis gives the sizes of the fragments in base pairs. The Y axis is the intensity of the luminescence given off from the fluorescent tag which is attached to the PCR primers which enables the machine to detect the DNA fragments.
If the PCR sample contains millions of DNA fragments, why would one sample only produce one data peak? 26

27. 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.

28. Here are Johanna’s data for three different DNA fragments from each of the meerkats
Martin!
Who is the daddy?
Malcolm
Martin 28

29. primer To make copies of the DNA you need to add to the PCR mix:
DNA polymerase
nucleotides of each base type.
primer

30. PCR uses a special DNA polymerase – Taq polymerase
Why? +
Where is Taq from?
What is one of the problems with using it?

31. 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.

32. PCR Summary

33. Once DNA has been amplified how is it searched?
DNA probes
Once DNA has been amplified how is it searched?

34. 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?

35. DNA probes Made of DNA Short Single stranded
Complementary to the sequence being searched for

36. 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.

37. 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.

38. 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.

39. 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.

40. 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.