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.
What is a reverse trasncriptase?
enzyme used to generate complementary DNA (cDNA) from an RNA template. Used by retroviruses
what is the aim of reverse transcription PCR?
To look at the expression of a gene of interest. A strong band means the gene is more highly expressed.
The other reason why you might want to do RT-PCR is to look at the length of the transcript produced. For example, if your variant is thought to interfere with a splice site, then you could predict that you’ll see a mRNA transcript of a different length.
What could be a potential problem with reverse transcription pcr?
differential expression could be due to the fact we started the PCR reaction with lots of RNA from brain, but only a small amount of RNA from the kidney sample for example
What is a housekeeping gene?
housekeepin gene is a gene that is expressed in all tissues at a similar level
Example of house keeping gene?
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase )
Beta-actin
How is housekeeping gene used in reverse transcription PCR?
The theory is that housekeeping genes are expressed at the same level in all tissues and so you’d expect to see a nice strong band in all samples, to suggest that we started with the same amount of RNA in all samples
Differences seen on the gel above should then be due to experimental variation in the amount of RNA loaded and can be corrected for.
How do we make RT-PCR quantative?
counting the number of copies of amplified DNA present.
We count the copies by using fluorescent molecules - “tags”.
How do you count the no. of amplifies molecules present in PCR?
Include a dye in the PCR reaction mix that fluoresces when it binds double-stranded DNA, e.g. an intercalating dye such as SYBR Green
Label a probe in the PCR that only fluoresces when it is incorporated in the PCR product, e.g. TaqMan
Microarrays for gene expression?
Lots of copies of the same probe in a spot
each spot gives the relative expression for one transcript
detects all known transcripts in one sample
What is proteomics?
arge-scale study of proteins
what is the proteome?
the entire set of proteins, produced or modified by an organism or system
why does protein variation occur?
Variant mRNA due to variation in splicing patterns
Post-translational modification. For example, glycosylation
Post-glycosylation modification. For example, sulfation
Exaplin steps of 2D Gel electrophoresis
A. Sample extraction and solubilisation
B. Isoelectric focusing using pH gradient in acrylamide gel
C. Re-solubilisation in SDS buffer; proteins negatively charged
D. SDS-PAGE separates proteins by molecular weight
E. Staining, quantification & analysis
What is HPLC?
High performance liquid chromatography
Used to separate, identify, and quantify each component in a mixture.
How does HPLC work?
Relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material.
Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.
What is mass spectrometry?
Technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio
Steps of mass spec?
1) inject sample
2) heater to vaporise sample
3) electron beam ionises sample
4) particles accelerated inot magnetic field
5) magnetic field sperates particles based on mass/charge ratio
what techniques are used in DNA analysis?
PCR, sequencing, microarrays
what techniques used in RNA analysis?
RT-PCR, qPCR, microarrays
what techniques used for proteomics?
2d gels, HPLC, mass spec
