1. copying & sequencing DNA
module 2 – biotechnology & gene technologies

2. From the spec

3. learning objectives Understand what the polymerase chain reaction is.
Understand what the PCR can be used for.
Understand how genes can be sequenced.

4. success criteria
Describe what happens in the polymerase chain reaction.
State what is required for the PCR to occur and explain why these components are important.
State advantages and disadvantages of the PCR.
Explain how fluorescently tagged nucleotides can be used to sequence genes.

5. starter
Draw and annotate a diagram to show how DNA replication occurs.
Include as much detail as you can – enzymes, primers etc…
Volunteer to do it on the board, if you’re brave…

6. part 1 copying DNA

7. polymerase chain reaction (PCR)
PCR is an method by which DNA can be replicated in the lab.
It can be used to create millions of copies of DNA in just a few hours.
It is essential in forensic science as very small samples of DNA are difficult to analyse.
This process amplifies DNA, so that it can be analysed.

8. what do you need?
A thermocycler – a machine that can quickly alter the temperature of the samples being amplified.
RNA primers provide the starting sequence for DNA replication. They also stop the two DNA strands from joining together.
DNA nucleotides containing the bases adenine, guanine, cytosine and thymine.
The enzyme - DNA polymerase.

9. Heat to 950C to separate the DNA strands
The Double Stranded DNA Molecule A G T C T C A G
Heat to 950C to separate the DNA strands

10. Cool to 550C to allow primers to bind (anneal) to DNA
DNA Strand A G T C C T
RNA Primers C T T C A G
DNA Strand
Cool to 550C to allow primers to bind (anneal) to DNA

11. G A G T C T C A G G C C G G A A G G T C A T C A G G
DNA Polymerase G
Original DNA strand A G T C T C A G G
Primer C C G
Nucleotides join on
Free DNA nucleotides
Nucleotides join on G A A G G T C A
Primer
Free DNA nucleotides T C A G G
Original DNA strand
Mix with DNA polymerase and free nucleotides and heat to 720C

12. a cyclic process
The whole process can be repeated many times, so the amount of DNA increases exponentially.
x2, x4, x8, x16, x32 etc.
Strand Separation
DNA heated at 95°C for 5mins
Binding of Primers
Mixture cooled to 55°C C
Mix with Primers (RNA strands)
DNA Synthesis
Mixture heated to 72°C
(optimum temp. for DNA polymerase)
REPEAT CYCLING
Mix with
Free Nucleotides
DNA Polymerase
With every cycle the amount
of DNA doubles
Suggest why DNA polymerase is referred to as a thermophilic enzyme.

13. Advantages & Disadvantages
It is a very rapid process – each cycle takes only minutes.
It does not require living cells – only viable DNA fragments.
It is useful when needing large amounts of DNA for genetic engineering.
Any contaminant DNA will also be amplified.
If a desired gene is to be copied, any ‘junk DNA’ either side will also be amplified.

14. part 2 sequencing DNA

15. Sanger Sequencing Method
Meet Frederick Sanger...
Biochemist
Cambridge University
English
Two Nobel Prizes
Still Alive
Sanger’s work in the 1970’s, which earned him his second Nobel Prize, involved the sequencing of DNA.
Sanger Sequencing Method
His method used modified nucleotides that do not allow another nucleotide to join after them in a sequence.

16. Introducing Sanger Sequencing
The method is based on the premature ending of DNA synthesis.
If modified nucleotides are used during DNA synthesis, the process can be halted.
What normally happens during DNA synthesis... T A T G G A T C T G A C C T T A G A T A C C T A G A C T G G A A T C
What happens if you modify a nucleotide...
You call these modified nucleotides, TERMINATORS T A T G G A T C A T A C C T A G A C T G G A A T C

17. what you need for sequencing
The reaction mixture for automated sequencing is very similar to that of PCR.
However, some of the nucleotides are fluorescently labelled, and if added to a growing chain, no further bases can be added.
What you need:
The DNA being sequenced.
A mixture of ‘normal’ nucleotides (A, T, C, D)
A primer.
DNA Polymerase.
Fluorescently labelled ‘terminator’ nucleotides. A T G C A C C

18. So what happens in each tube?
Lets take the example of the tube with an adenine terminator G T C A
Now let’s imagine this is the sequence of the unknown DNA strand:
C C G T C T A G C A C T C A A G C T C T
What are the possible terminated sequences going to be when the reaction is over?
G G C A
Because there are both ‘normal’ and ‘terminator’ nucleotides in the mixture, there is a chance that either is placed as the next base
G G C A G A
G G C A G A T C G T G A
G G C A G A T C G T G A G T T C G A
G G C A G A T C G T G A G T T C G A G A

19. Remember that this is happening in four test-tubes, each with a different type of terminator nucleotide. DNA fragments in each of the four tubes are going to be of varying lengths. Now the lengths of DNA need to be separated, so that we can see why we went through all of this trouble...

20. Back to Sanger Sequencing
The fragments produced during the reactions can be separated using gel electrophoresis.
The smallest fragments will move furthest along the gel in a fixed period of time.
Due to being radioactively labelled, we can see where the DNA fragments end up, by placing photographic film over the gel, after the run.
Terminator C
Terminator A
Terminator T
Terminator G

21. Modern Sequencing - what the fluorescent bases do
If a modified (terminator) nucleotide is added, the DNA polymerase is ‘thrown off’ and can no longer be completed.
It will be shorter than a fully replicated molecule.
What normally happens during DNA synthesis... T A T G G A T C T G A C C T T A G A T A C C T A G A C T G G A A T C
What happens if you modify a nucleotide...
Remember that these modified nucleotides are fluorescently labelled. T A T G G A T C A T A C C T A G A C T G G A A T C

22. Molecules of every possible lengths would be made…
4 bases, 4 colours
As there are 4 different bases, each type of modified nucleotide has to have an individual fluorescent marker.
As the reaction proceeds (and modified nucleotides halt individual reactions), varying lengths of DNA are made:
Adenine
Thymine
Cytosine
Guanine
T A G T C A G
T A G T C A G T A A T
T A G T C A G T A A T A G C T A T
T A G T C A G T A A T A G C T A T A C
T A G T C A G T A A T A G C T A T A C G A T A G T
T A G T C A G T A A T A G C T A T A T G A T A G T A T A A
T A G T C A G T A A T A G C T A T A T G A T A G T A T A A T A
Molecules of every possible lengths would be made…

23. spot the colour
The strands are separated according to size in a process similar to gel electrophoresis.
A computer analyses the order of fluorescent markers,
and can therefore deduce the order of bases in the
template DNA strand.
Using the key below, work out the order of bases on the
template DNA strand: A T C G

24. Plenary
Explain how the PCR enables forensic scientists to analyse minute samples of DNA found at the scenes of crime.
Suggest why DNA polymerase used in the PCR is obtained from thermophilic bacteria.
Homework
Stretch and Challenge section on page 171 of textbook.

25. learning objectives Understand what the polymerase chain reaction is.
Understand what the PCR can be used for.
Understand how genes can be sequenced.

26. success criteria
Describe what happens in the polymerase chain reaction.
State what is required for the PCR to occur and explain why these components are important.
State advantages and disadvantages of the PCR.
Explain how fluorescently tagged nucleotides can be used to sequence genes.

27. Locating and sequencing genes
DNA probes
Using BAC gene library
DNA sequencing
Gel Electrophoresis
PCR

28. Flow diagram: Sequencing the Genome
Use resources (p184-8 green book)
Restriction enzymes
BAC
Growth of bacteria
Extraction and RE
Sequencing
Computer processing