Basic Molecular and MLH1

Question 1

How many SNPs are there in the human genome?

Answer 1

Approx 10 million

Question 2

Roughly what percentage of the human nuclear genome is protein coding DNA?

Answer 2

Approx 1%

Question 3

Briefly describe what transcription and translation is.

Answer 3

DNA acts as template for formation of mRNA - transcription

Introns are spliced out of mRNA

mRNA moves into cytoplasm where translation will take place

mRNA template is read by a ribosome which moves along the strand reading the sequence.

tRNA is also in the cytoplasm and has attached to it an amino acid (there are 20 amino acids)

The tRNA which is carrying the correct amino acid will then attach to the ribosome and release its amino acid into the growing chain - this chain is what produces the protein

Translation - ribosomal RNA translates the mRNA onto a protein

Question 4

Basically describe PCR….

what components does it require?

Answer 4

Exponential replication of DNA

DNA is denatured at high temp
Temperature is dropped to allow primers to anneal to template (temp is determined by the melting temp of the primers)
Extension - taq polymerase is active and begins extension of the strand from the primer ~70c

DNA template
Primers specific for the region you want to amplify
Taq polymerase - binds next to primers and extends sequence by adding bases
dNTPs - G, T, C and A
Thermal cycler

Question 5

How does the EZ1 extraction system work (basic)?

Answer 5

• magnetic silica bead method
• uses presence of chaotrophic salt to lyse cells and denature proteins
• DNA binds to beads and a series of wash steps remove all the cell debris etc.
• extracted DNA is then elated into water or TE buffer.

Question 6

?why can salt inhibit PCR?

Answer 6

?inteferes with and disrupts polymerase action?

?Interacts with the magnesium ions which DNA polymerase requires?

Question 7

What is the structure of DNA?

Answer 7

• nucleotide bases which pair together
• its well known shape is due to it coiling together with a sugar phosphate backbone
• strands held together by hydrogen bonds

Question 8

What is DNA methylation?

Answer 8

• epigenetic mechanism
• addition of a methyl group onto a ‘C’ nucleotide
• a method of changing the activity of a segment/region of DNA without changing the actual sequence

Question 9

If DNA is methylated what does it mean?

Answer 9

DNA methylation typically represses transcription of the region/gene.

Question 10

What is the promoter region?

Answer 10

The transcription start site.

Question 11

How is a DNA methylated?

Answer 11

Transfer of a methyl group onto the 5th carbon of a ‘C’ nucleotide.

Forms 5-methylcytosine.

Occurs at ‘C’ bases which immediately preceed a ‘G’ base e.g. CpG islands

Question 12

Are CpG islands usually methylated or not?

Answer 12

About 80% are methylated - however, of those unmethylated CpGs the majority are in promoter regions.

Question 13

What happens when methylation occurs in the promoter of a critical gene?

Answer 13

It can lead to tumorigenesis.

Question 14

What is MLH1?

Answer 14

Gene involved in DNA repair.

Question 15

How can we determine if a region of DNA is methylated?

Answer 15

We can do Bisulfite Genomic Sequencing - gold standard technique.

Question 16

What is the basis of Bisulfite Sequencing?

Answer 16

• based on the fact that methylated V unmethylated Cytosine bases react differently to the presence of Sodium Bisulfite

Question 17

What does Sodium Bisulfite do to cytosine?

Answer 17

• when it is unmethylated it is converted into a Uracil residue
• in subsequent PCR reactions this then becomes a Thymine residue
• methylated cytosine bases are protected from this conversion (actually it just happens much slower)

Subsequent sequencing can then differentiate between C or T at each base we are interested in.

Question 18

What is specific about the primers for this test?

Answer 18

• the reverse primer is biotinylated,

- the primers are complementary to the Bisulfite converted sequence only.

Question 19

How does Pyrosequencing work?

Answer 19

• the prep steps for Pyro basically expose the PCR products to streptavidin beads which bind to the biotinylated primer
• the DNA is now attached to the bead
• we denature the DNA and wash away everything else
• we then add our sequencing primer which s complementary to the region immediately prior to our region of interest
• we then load a cartridge with the enzyme, substrate and nucleotide bases that are needed for the reaction
• the machine starts the reaction and release nucleotide bases into the reaction in a predetermined order called a dispensation order
• each time a base is incorporated, light is released and detected by the camera
• the amount of light is proportional to the number of nucleotides added
• after each base, apyrase is released to clear any unused ones away and then the next base is added and the reaction continues
• gives you a real time information as the computer plots the peaks as soon as the light is detected

Question 20

What is the dispensation order?

Answer 20

• the order in which you tell the machine to release the nucleotides
• usually specific to what sequence you’re expecting however can also ask it to dispense a nucleotide that you’re not expecting to be there - as kind of a ‘control’
• can also ask it to dispense duplicate bases so that you know a consecutive run of multiples of the same base has been fully ‘read’

Question 21

How do you know that the Bisulfite Conversion is fully complete?

Answer 21

• the region of interest contains a ‘C’ nucleotide which is known to always be unmethylated
• this base will always convert to a ‘T’ during Bisulfite conversion
• the software knows to expect this and to check this it dispenses ‘T’ then ‘C’ then ‘T’
• if there are any ‘C’ residues remaining here it suggests that the conversion hasn’t fully completed
• the software will flag a warning if more than 5% bases are unconverted
• it will fail the replicate if >7% of bases are unconverted

Question 22

Did you get many Bisulfite warnings during the validation?

Answer 22

• quite a few warnings were obtained for the control DNA
• we wonder whether this is due to repeat freeze thaw? - so will aliquot into smaller sizes in future - haven’t had enough samples to investigate this yet
• compared data of replicates with a warning to those without e.g. range and mean of % methylation at each island and didn’t find a difference
• havn’t really had an issue with the patient samples and especially not with ones that have been extracted from an FFPE slice

Question 23

Briefly, what is the Pyrosequencing reaction?

Answer 23

Base incorporated = pyrophosphate released.

Cascade is then initiated which leads to release of an amount of light which is proportional to the number of nucleotides added (up to a point) - visualised as a peak on the pyrogram.

Apyrase degrades left over bases and the next one is then added, cycle repeats.

Question 24

What are the drawbacks of Pyro?

Answer 24

• only good for relatively short reads as cartridge can only be filled with so many reagents which would be used up,
• although the peak is proportional to the amount of light the more bases there are the less accurate it becomes e.g. an tell the difference between 1 or 2 bases but not between 4 v 5,
• generally uses all your product up, if it fails you have to start over!
• for the MLH1 assay you would have to go back to PCR1 products and set-up PCR2 again.

Question 25

Why is the PCR nested?

Answer 25

• not enough product obtained from single round of PCR
• assume this is because DNA is being extracted from FFPE which already yields fragmented DNA
• this is then coupled with harsh bisulfite conversion process.