6 - Molecular Diagnostics Flashcards
How was DNA sequenced in the 1970’s and 1980’s?
You had to clone the DNA, prep the plasmid from a culture, isolate the fragment from the vector, fun the sequencing reactions, and then fun the products on a gel.
Then transfer the gel from glass plates to filter paper without ripping. Dry them, radiograph and read by hand. Error prone reading.
How did DNA sequencing change in the 1980’s and 1990’s?
PCR and fluorescent labeling rather than radioactive.
Could read 4x as much per gel and didn’t need to clone segments because PCR could amplify target.
Optical detection allowed longer electrophoresis, which 4x the read lengths.
What was a benefit of capillary electrophoresis?
Faster separation and greater parallel processing capacity.
Overcame trouble of high GC content.
What the characteristics of next-generation sequencing?
Adaptor ligation step allowed multiple samples to be analyzed together.
Eliminated physical separation of molecules by electrophoresis.
Reactions performed in situ on solid substrate.
What are some limitations to next-generation sequencing?
Read lengths are much shorter, but the number of parallel reactions is much greater.
Net effect: increase capacity ~10^6
What is single nucleotide polymorphism? What makes something polymorphic by definition?
Genetic variation when a different base is present than most commonly found.
A site is polymorphic if a minor allele is present at a population freq of >1%.
What is a variant called when it is found at a frequency of < 1% in a population?
A SNP, and the locus is not considered polymorphic.
What are two ways that SNPs can be assayed?
using RT-PCR or genome-wide genotyping arrays.
How is an RT-PCR done?
Allele specific probes with fluorescent dye and a quencher bind to target sequence during annealing phase.
During elongation, quencher and dye are separated yielding a signal.
If the reactions contains both alleles, and each probe has a different colored signal, 2 colors will be generated in equal amts.
How does a SNP genotyping array work?
Still uses allele specific hybridization, but on a solid substrate with coordinates of each SNP pre-selected.
What are some limitations to the SNP assays?
Limited to specific, previously identified base; incapable of finding variation at other locations (even within same gene).
Unable to detect deletions, as a null allele will appear to be homozygous for the remaining allele.
How does melting-based genotyping work?
Mismatched heteroduplexes will melt at lower temps than AT bonds.
Partly denatured DNA migrates differently than single or dsDNA. By separating molecules near melting point, species can be distinguished.
What are the advantages and disadvantages of melting-based genotyping?
It can find SNPS and indels in a small region of DNA, but not at a specific position. It’s rapid and relatively inexpensive.
Can’t specify exact variant and can’t distinguish functionally important variants from silent ones. Also insensitive to deletions.
What are indels? How can they be found?
Insertion or deletion variants.
Found by comparative genomic hybridization.
What are micro-satellites?
Small indel that consists of a tandemly repeated array of a very short repeated sequence.
For most microsatelite loci, the copy number of the repeated sequence turns out to be polymorphic within a pop, but stable from generation to generation to be usable as a mendelian trait.
