Advanced Molecular Techniques

Question 1

Name 2 techniques to analyse DNA at the nucleotide level

Answer 1

DNA sequencing

PCR plus restriction analysis

Question 2

Name 5 techniques to analyse DNA at the gene level

Answer 2

Southern hybridisation
Northern hybridisation
RT-PCR
Microarray
DNA fingerprinting/DNA profiling

Question 3

Name 3 techniques to analyse DNA at the chromosome level

Answer 3

Karyotyping
FISH
Chromosome painting

Question 4

Who would be interested in genome information? What ethical issues arise from this?

Answer 4

Family 
Potential spouse 
Doctors 
Government 
Police 
Schools 
Insurance companies
Can the knowledge help prevent illness later in life? 
Does it open up areas for discrimination? 
Who owns DNA sequence?

Question 5

What are allele specific primers?

Answer 5

Primers complementary to specific alleles; will hence bind tighter to them
E.g. In sickle cell A>T mutation, sickle specific primer therefore has A rather than T
Only get PCR product if right primer used

Question 6

Describe reverse transcriptase PCR

Answer 6

Recognises RNA - makes a copy ofDNA from RNA (cDNA)
SsDNA made from rna 
Primer of Ts add RT
Makes copy of mRNA
Can degrade rna but not dna with rnase
SsDNA -
Design 2 primers (forward and reverse)
Made strand double (adding onto primer)
PCR
Look at gene expression

Question 7

Describe microarray technology

Answer 7

Pattern of lots of dna on a piece of glass/plastic called gene chip
Analyse 1000s of genes at the same time
Each block has 100s of bits of dna
Results in lots of different dots - each is a bit of DNA

Question 8

Describe using microarray technology to observe conditional gene expression

Answer 8

Make cDNA with RT
Cancer and normal given different colours
2 pools of CDA - different colour labels
Combine
Hybridise with array containing all human genome
Gene switch on in cancer - lots of red cDNA
If this gene is switched off in normal - no green
All completely red when hybridised are genes switched on in cancer cell but not normal
Completely green = switched on in normal but not in cancer
Equal = on in both
Can look at all genes at the same time - can see which are more or less expressed in cancer cell
Either can be red or green

Question 9

Describe Array Comparative Genome Hybridisation (microarray technology)

Answer 9

DNA extracted from cells from patient and cells from normal controlLabelled with 2 different fluorocarbons
Mixed in equal quantities
Hybridise to microarray of clones
Read red and green fluorescence
Work out red to green ratio for each cell, align to database of clones
Red spot = more dna from patient compared to normal - this bit of dna is duplicated - duplication that shouldn’t be there
2 healthy individuals = should be equal colours
Red spots = duplications
Green spots = patient is missing part of DNA

Question 10

What is the basis of DNA fingerprinting?

Answer 10

Minisatellites repeated over again
Different sizes of fragments
Individuals have different repeats of same region
Unique patterns using dna

Question 11

What can DNA infer printing be used to show?

Answer 11

Family relationships, i.e. Children will have same repeats as parents- some from each parent

Question 12

Describe FISH (fluorescent in situ hybridisation)

Answer 12

Fluorescent bit of dna

Hybridise it
Denature dna 
Renature
Probe will hybridise where it has sequence identity (rom heteroduplex)
At that point - fluorescent tag

Question 13

Describe denaturing and renaturing DNA

Answer 13

When a double-stranded molecule of DNA (dsDNA) is heated (or treated with an alkaline solution) it is said to denature. That means the hydrogen bonds between the bases are broken and single-stranded DNA (ssDNA) is released. If we subsequently cool the mixture containing the ssDNA then these can come back together – we say renature or anneal – to form the dsDNA molecule because the hydrogen bonds reform. The molecule is able to reform as the 2 strands have complementarity.

Question 14

Describe DNA hybridisation

Answer 14

Now lets denature the same piece of dsDNA again. Before allowing the mixture to cool we add another piece of ssDNA that has an identical sequence to one of the strands. The only difference is that we have labelled this molecule with a radioactive (or fluorescent) marker. When the ssDNA reanneals some of the molecules will do so with the labelled piece of DNA. It is now possible to identify this labelled DNA using photographic film. This forms the basis for molecular hybridisation which is used in many molecular techniques

Question 15

What is southern blotting?

Answer 15

There are several different variant of hybridisation techniques: 1. Southern blotting – named after it’s inventor Prof Sir Ed Southern this uses DN!
probes to identify complementary DNA sequences after gel electrophoresis.

Question 16

What is northern blotting?

Answer 16

Northern blotting – a molecular biochemist’s joke here! Uses DNA to detect RNA species in a similar way to the above.

Question 17

Describe southern blotting briefly

Answer 17

Southern blotting involves an initial DNA gel electrophoresis step to separate fragments of DNA followed by the subsequent transfer to a membrane hybridisation of a probe to detect a specific piece of DNA and visualisation of the labelled probe

Question 18

What are the main steps in southern blotting?

Answer 18

Digest genomic DNA with restriction enzymes
Separate DNA fragments by gel electrophoresis
Transfer DNA fragments to nylon
Hybridise filter with labelled gene probe
Detect hybridisation (and hence DNA of interest) by exposure of filter to X-ray film

Question 19

What is southern blotting usually used for

Answer 19

Usually Southern blotting will be used to analyse restriction fragments of DNA – this can from genomic DNA or from more simple sources like a cloned gene or PCR product.

Question 20

How is a southern blot performed?

Answer 20

So how do we perform a Southern blot? After separating DNA on a gel the fragments of DNA need to be transferred to a solid support – usually something like a nylon or nitrocellulose filter is used. The gel is soaked in an alkaline solution which denatures the DNA into ssDNA molecules and these are transferred onto the membrane. A simple way to do this is where DNA is simply blotted by capillary action for the gel onto the filter. An alternative method is to use electrophoretic transfer – a voltage is applied across the set-up such that negatively charged DNA moves from the gel onto the filter.

Question 21

What happens in southern blotting once DNA has been transferred to nylon filter?

Answer 21

Once the DNA has been transferred on to the nylon filter the gel can be discarded. It is important to recognise that the DNA on the filter is now in the single-stranded form.
The filter is now placed in a solution containing the labelled DNA probe. This can be formed from a cloned piece of DNA that we already have available or can be produced in the lab using an oligonucleotide synthesiser – a machine which produces short stretches of DNA from dNTPs with whatever sequence we programme into the machine. The labelled probe will float freely in solution and will bind wherever it finds ssDNA on the filter with a complementary sequence. After binding the probe we wash the filter to remove any unbound probe and the detect binding using photographic film or fluorescent detection methods.

Question 22

Why use southern hybridisation

Answer 22

Investigate gene structure e.g. Large deletions or duplications
Investigate gene expansions, triplet repeats e.g. Huntingtons
Investigate mutations in genetic tests using allele specific problems e.g. Sickle cell disease
Investigate variation, genetic relationships e.g. DNA fingerprinting

Why do we use these methods. Southern blotting allows us detect pieces of DNA from complex mixtures that would otherwise be very difficult to detect. It also allows us to detect very small amounts of DNA that may not be visible by staining of DNA in a gel. In many cases we can use Southern blotting in combination with PCR to detect things like gene structure, gene expansions and repeats, mutations and genetic variation etc.

Question 23

What are characteristics of DNA probes in blotting?

Answer 23

Probes do not have 100% complementarity to the sequence we are using them to detect
Probes do not have to completely align with the sequence they are being used to detect
Probes do not affect the position of the target sequence on a gel

Question 24

What is a dNTP

Answer 24

Deoxynuclotide triphosphate (can be used in DNA replication)

Question 25

What is ddNTP?

Answer 25

Dideoxynucleotide triphosphate - contains a H at the 3’ end meaning that it blocks further elongation of DNA once incorporated as no further phosphodiester bonds can form

Question 26

Which direction does DNA replication occur?q

Answer 26

5’ to 3’ (i.e. New nucleotides added at 3’ end)

Question 27

What happens when ddNTP is added? (Chain termination - Sanger sequencing)

Answer 27

Add a DNA template with free dATP, dCTP, cGTP, dTTP and ONE of the ddNTPs and incubate at 37C
There is a chance that the ddNTP used may bind instead of its respective dNTP
Because we have lots and lots of the template over time we will see a mixture of new DNA molecules produced of different length depending on where the ddNTP is incorporated.

Question 28

Describe Sanger sequencing

Answer 28

In the Sanger method 4 separate tubes are used each with a unique ddNTP and a labelled primer to initiate DNA synthesis. After incubation the products of the reactions are run out on a gel using separate lanes for each of the reaction tubes. This will separate the labelled fragments out on the basis of size. We are then able read off the sequence from the bottom of the gel to work out the nucleotide sequence in the newly synthesised strand.

Question 29

What is fluroescent dideoxy DNA sequencing?

Answer 29

In reality, although we rely on the same basic Sanger method we now use fluorescently labelled ddNTPs and use them all together in the same tube. The different length fragments are then separated on a very thin capillary gel and as the fragments fall of the end they are detected by a laser. In this case A= green, T= red, C= blue, G=black. We can produce this as a chromatogram (seen top right above) from which we can read the sequence directly.