Biology 3594A Study Guide - Final Guide: Amplicon, Fetus, Prenatal Diagnosis
13 May 2018
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Lecture 19 Flashcards
Front (Term)
Back (Definition)
True negative vs false
negative
True negative: biologically what you assayed is just not there
False negative: the thing is there but you failed to detect it
if a PCR reaction has no
amplicon is this sufficient
evidence that the target was
not present?
NO not sufficient evidence for a true negative
What steps would increase
confidence for the absence of
the target?
- more negative and positive controls - different technique (validation?)
- could sequence to check for inversions and mutations that might have
resulted in the target not being amplified
Need reproducibility
Should a clinical test be
based on a negative result
(the absence of a product)?
NO
Clinical test should have high resolution, reproducible product
Validated by studies with large sample size and the same results
New applicationsof
CRISPR/Cas 9 system on
mutant DNA detection
- ForeignDNA
- Cell free DNA = cfDNA
- FetalDNA in mother's plasma (could sequence DNA and learn about
disease or sex of fetus in first trimester)
- TumorDNA (after treatment is any tumor DNA still there?)
- Circulatingtumor DNA = ctDNA (gather the DNA from destroyed cells
from chemo?)
- Minimalresidual disease
- Somaticmosaicism
- Germlinemosaicism
- Microbialinfection/contamination
- ContaminatingDNA
find more resources at oneclass.com
find more resources at oneclass.com
Why looking for mutant DNA
is important
Mutant DNA is a biomarker for cancer and prenatal diagnosis
- could do a noninvasive diagnosis
- individualized medicine
Why looking for mutant DNA
in individuals is hard
Mutant DNA is in very low abundance
- current assays aren't very good at finding it, they lack sensitivity even
if they are low cost or simple
How CRISPR/Cas 9 enriches
low abundance DNA
CRISPR/Cas 9 is used in DNA editing
- it cuts very specific things
Cut all the WT DNA so all that is left is the mutant
- assay is not about discovering new mutations, but for finding a
mutation you already know about
- known disorders and diseases
Goal for CRISPR/Cas9 assay
sensitivity
0.01 - 0.1%
The levels of cfDNA, ctDNA and mosaicism
AFLP
- what it stands for
- what it's used for
Amplified Fragment Length Polymorphism
- used to detect alleles that differ in sequence length
- so used when WT and MU have different sequence lengths (i.e. when
MU is a deletion or duplication)
- NOT FOR BASE SUBSTITUTIONS
Resolution of AFLP and
methods used to achieve it
(3)
Single nt resolution!!
1. High % Polyacrylamide gel electrophoresis (PAGE)
- smaller than agarose gel
2. Silver stain
- more sensitive than ethidium bromide in detecting ds DNA
- very sensitive for length difference detections
- based on reduction of silver cations to insoluble silver metal by nucleic
acids
3. Capillary electrophoresis
- tiny tubes containing compounds that can slow migration to tell single
nt differences
- electrophoresce DNA through the tube
Abundance detection of AFLP
5% abundance of mutant in a mixture of MU and WT, so not quite
where we want to be for cell free DNA
NEPB PCR
- what it stands for
Non-extendable probe blocker PCR
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
True negative: biologically what you assayed is just not there. False negative: the thing is there but you failed to detect it. No not sufficient evidence for a true negative. More negative and positive controls - different technique (validation?) Could sequence to check for inversions and mutations that might have resulted in the target not being amplified. Clinical test should have high resolution, reproducible product. Validated by studies with large sample size and the same results. Fetaldna in mother"s plasma (could sequence dna and learn about disease or sex of fetus in first trimester) Tumordna (after treatment is any tumor dna still there?) Circulatingtumor dna = ctdna (gather the dna from destroyed cells from chemo?) Mutant dna is a biomarker for cancer and prenatal diagnosis. Why looking for mutant dna in individuals is hard. Current assays aren"t very good at finding it, they lack sensitivity even if they are low cost or simple.
