Techiques Flashcards
Peptide nucleic acid FISH
PNA against an organism-specific ribosomal RNA which is conjugated to a fluorophore
PCR ESI-MS
PCR electronspray ionization-mass spec
Starts with PCR amplification of organism-specific 16S ribosomal RNAs - then those amplicons are submitted directly for mass spec after nucleic acid purification.
Works very well as long as organisms differ in 16S rRNA sequence.
Steps for karyotyping
Isolated live cells
Culture
Treat cells with colchicine
Put cells in a hypotonic solution to cause cell swelling, spreading the chromosomes out
Fix in methanol-acetic acid buffer
Splat the cells to spread the metaphase chromosomes on a glass slide
Traditional stain for chromosomes
Giemsa
The “G” in “G-banding” actually stands for Giemsa.
Resolution of karyotype
3-5 megabases
ie, 3,000,000-5,000,000 bases
Approximate human genome size
3 x 10^9 bases
Why do we use a 5 micron section for FISH?
5 microns is approximately the size of the average cell, so you are getting roughly a monolayer
Why are G’s a problem with NGS?
When you use two color encoding:
T = green
C = red
A = green + red
G = nothing
So, towards the end of the read, you will get serial “G’s”
Confidence strand
A = 65
E = 101
Numbers are in ASCII code to match one character to one character
Thread quality
20 = 1% error rate
30 = 1 per 1,000
40 = 1 per 10,000
Array CHG
Array comparative genomic hybridization
Take red-labeled patient DNA and green-labeled control DNA, then that DNA is applied to an array of wells with probes that represent spaced on genome sequences.
Based on the relative normalized intensity in each well of the array, you can then determine whether there has been a gain or loss in the patient DNA compared to the control DNA.
It’s like FISH in reverse – the probe is in situ and the DNA is mobilized.
Array CGH readout
Each well is expressed as log2(R/G), such that no difference = log2(1/1) = 0.
We define a “significant difference” as having a value greater than 0.25 or less than -0.25, but really we still wouldn’t believe a single point elevation. Rather, we would want to see a difference across a range of positions (usually at least 5) which are adjacent to one another in the chromosomal sequence.
Resolution of aCGH
10’s of kbs (at its best, when wells represent 1,000 bp spacing)
What does aCGH miss that karyotype and FISH can see?
Translocations and inversions
Polyploidy (3n genome rather than 2n, as in a partial hydatidiform mole)
Otherwise copy number neutral mutations
Two flavors of uniparental disomy
Isodisomy: A region of a chromosome inherited from mom is lost, but is replaced by a copy of the same region from dad’s chromosome. This produces a copy number neutral karyotype, but with homozygosity within this region.
Heterodisomy: A region of a chromosome inherited from mom is lost, and is replaced by a copy of another region of dad’s DNA. This produces a copy number gain at one site and loss at another.
When does cold ischemic time factor in to PCR amplification?
Even 24 hours of cold ischemic time does not affect the success rate of PCR amplification.
When does cold ischemic time factor in to RNA quatitation?
Up to 12 hours out has been shown to still be effective for analysis, but less than 12 hours is generally recommended.
As a matter of quality, the goal for ALL specimens where molecular techniques will be employed should be to have a cold ischemic time of. . .
. . . less than 1 hour
Effects of formalin fixation on DNA
Can lead to shorter average DNA fragments and cause random cytosine deamination
Pyrosequencing
Sequencing-by-synthesis method
Utilizes a series of reactions to measure the release of inorganic pyrophosphate as each nucleotide is incorporated into the DNA chain
Melting curve analysis
Detection of mutations by measuring the changes in fluorescence generated by different dissociation curves between wild-type and mutated samples.
Used to detect single base pair substitutions and small indels. Can also be used to detect methylation status of a specific region.
Fast, close-ended system that is not labor intensive.
Importantly, this assay detects that a mutation is present, but now what mutation is present.
Sensitive to a VAF of around 10%.
Methylation sequencing
NGS with the ability to read 5-methylcytosine
“Generations” of NGS
“Ion torrent” sequencing or “semiconductor” sequencing
Detects small changes in pH from H+ released by nucleotide incorporation.
The alternative to pyrosequencing for sequencing by synthesis and reading byproducts of nucleotide incorporation.
Comparison of the major NGS platforms’ strengths and weaknesses
