Enabling Technologies Flashcards
What is PCR?
Exponential amplification of a DNA fragment of known sequence
Components of PCR and their functions?
Template – DNA to amplify
Primers – Short pieces of ssDNA (15-30bp)
Polymerase – thermostable enzyme (Taq)
Nucleotides – single base mixture (dNTPs)
Buffer – To maintain pH
MgCl2 – Essential for polymerase activity
3 different incubation temperatures and what do they result in?
94 deg c = denaturation
60 deg c = annealing
72 deg c = extension
Name a type of DNA sequencing?
Sanger (dideoxy sequencing)
General steps of sanger sequencing?
1) DNA isolation & amplification
2) Sequencing reaction
a. Strand separation
b. Anneal primer
c. Extension
d. Termination
3) Separate fragments and determine sequence
1) DNA isolation & amplification
DNA should be relatively pure - free of protein and cellular debris, especially
DNases
Proteases
PCR inhibitors – Haem-containing compounds
Amplification by PCR, or cloning, to give sufficient identical copies of the region to be sequenced
2) a. Strand Seperation?
1) Heat DNA to break H bonds between 2 stands, seperating them.
2) aim to sequence template strand
2) b. Anneal Primer
Aim to anneal the sequencing primer which is designed to be complimentary to the template strand.
In the extension mix, add some nuclotides.
Dna Polymerase goes along template strand and added complementary bases.
2) b. What is purposed of ddNTP’s?
dideoxy nucleotides:
1. Dye attached, different dye on each type of nucleotide
- lack the 3’hydroxyl group needed for chain extension – its used to make the phosphodiester backbone
What happens when a ddNTP is added onto the template strand?
the polymerase can’t add any more bases because of the lack of a 3’ hydroxyl group=termination
What sort of mixture do you end up with?
So what we end up with is a reaction mixture where the double stranded DNA molecules are all different lengths.
Sometimes the primer will be extended by only a few bases before the polymerase incorporates a dideoxy nucleotide which stops the extension reaction.
Other times the polymerase will trundle along for ages, incorporating dNTPs before it finally incorporates a dideoxy nucleotide.
3) Seperate fragments and determine sequence
After seq reaction, end up with lots of labelled molecules.
Denature the newly synthesised DNA - giving us a mixture of free labelled strands and free unlabelled strands.
Then load the fragments onto gel-filled capillary (same as agarose)
Apply electric current across gel. -ve charged fragments move down gel towards +ve electrode, smaller fragments move more quickly
3) what is the purpose of the laser beam?
As the fragments come off the end of the gel they pass through this laser beam which excites the florescent dye attached to the dideoxy at the end of each strand
This causes the dye to fluoresce and that florescence is detected by this photocell.
It’s detected as a “peak” of florescence as each fragment passes through the laser beam
What are fluorescent peaks recorded on?
electropherogram
What is a microarray?
An ordered assembly of nucleic acids immobilised on a solid support.
Support – glass – similar to microscope slide
What are SNP microarrays?
microarrays that hybridise adjacent to SNPs, rather than to RNA transcripts.
How are SNPs detected in SNP microarrays
SNP is extended by one base that is fluorescently labelled and detected using a high defintion scanner
Whats does one spot in the SNP microarray contain?
Lots of copies of the same SS oligonucleotide (a probe).
Each probe is for genotyping one SNP. Designed to hybrisdise with one SNP.
Explain the mechanism of SNP microsarray briefly?
We take that DNA sequence and we design a probe complementary to the region next to the SNP.
We then take that probe and attach it to a glass slide.
But not just one copy, lots of copies of the same probe, all being stuck in a spot on the slide.
And we do the same thing for the next SNP we’re interested in
And we do that for all the SNPs we’re interested in.
We then take DNA from our patient and wash it over the slide
It will then stick, or hybridise, to its complementary probe.
what kinds of study is permitted due to SNP microarrays?
GWAS (genome wide association studies)
What is transcriptomics?
the study of transcriptomes
what is a tracriptome?
The transcriptome is the set of all messenger RNA molecules in one cell or a population of cells
What is reverse transcription PCR?
Technique commonly used in molecular biology to detect RNA expression
how is reverse transcription PCR used?
using the enzyme reverse transcriptase to convert RNA into cDNA and perform PCR on the cDNA and run products out on a gel.
