DD6

Examples of mutations PCR can detect?

• Point mutations / SNPs

• Deletions

• Insertions

What do PCR methods rely on?

eaction products being of different sizes in the absence or presence of a mutation, or the reaction not working at all when using a mutated template

How can PCR be used to detect changes in EGFR TKI?

by designing primers to give different sized PCR products in the absence and presence of the mutation

Describe the method of detecting mutations using PCR?

In the wild type the restriction enzyme will cleave the sequence and create over hang ends

Mutation does not recognise the sequence there for does not cleave

Leaving full length DNA strand

Deletion and insertions will change the size of the products

Describe Allele-Specific PCR (ASPCR)?

No change to restriction sites

3 prime end if not complementary to mutant only to wild type

DNA polymerase does not bind and do not get amplification of that DNA

How can qPCR be used to detect mutations?

Two fluorescent TaqMan probes – one specific for wild-type sequence, the other for the mutation

Each have own colour

So when amplified detect which one via the colour or a combination

Describe microarrays?

are microchips or flow cells containing many microscopic spots of different DNA or RNA probes → oligonucleotides that will bind to complementary DNA in the sample

What are microarrays used for?

assess expression of large numbers genes, determine genotype or sequence nucleic acids

What are the applications of microarrays?

• Basic research, including target identification

• Human diagnostics

• Personalized medicines

Describe how microarrays can be used to detect gene expression?

DNA is labelled whatever binds will show its colour if expressed and bind do is not washed

How can RNA be used in microarrays?

• Can via reverse transcriptase

• Can be reverse transcriptase and PCR to amplify it up

Describe microchip technologies?

• Genomic DNA is taken and digested with different types of restriction enzymes into fragments

• Adaptors stick to the ends → like PCR primers

  • All have the same sequence on them

• Can be amplified by PCR with primers that recognise these ends

• These are then digested into smaller fragments of around 20 nucleotides and labelled with fluorescent tag

• The loaded on chip

Define copy number variations?

• Short sequences of DNA

• That can be within genes or can be extra genomic

• In both coding and non coding regions

• Have different alleles → different numbers of copies

How are forensics and identification done?

as all have different numbers an copies of these specific copy number variations

What is the BRCA gene type?

autosomal dominant

What was the first generation of DNA sequencing?

Sanger

Chain terminators

Each label with a different fluorophore

Result is end labelled sequences of different lengths

What is 2nd generation sequencing (NGS)

Microarray Sequencing Technology allowing for more rapid sequencing to be done

Describe NGS?

• Millions of clusters, each with around 1,000 molecules of DNA can be sequenced in a matter of hours

• Systems tend to be flow-based

• Sequential processes and imaging

Describe the illumina sequencing?

each nucleotide coloured

when imaged the colour of the dot represents a nucleotide

fluorescent molecule and this terminating group on the end that both cleaved off

Next nucleotide added by DNA polymerase and another fluorescent

Now only second one fluorescent

Repeats

Describe real time sequencing technologies?

Nano wells

• DNA polymerase sitting at the bottom of each well

• Can detect fluorescence at each of these wells

• DNA passing through the polymerase and the DNA is adding one nucleotide at a time

• Each nucleotide has a fluorescent probe

• Which can be detected and responds to a specific nucleotide

• Same time DNA end group is cleaved off so it is washed away

• Then adds the next nucleotide and so one