AMT FINAL Flashcards
what are the 3 types of dyes used to dye gels?
ethidium bromide, SYBR green, gel red
A good standard curve must have the following properties:
-it must be a straight line
-usually a minimum of 5 points is ideal
-for 100% PCR efficiency, the slope will be -3.3
-correlation coefficient should be >0.9
what is the ideal GC content when designing primers?
50-60%
denaturant, lowers denaturing temp
-used with high secondary structure (GC-rich DNA)
formamide
what happens if you increase salt in a PCR reaction?
-slows denaturation of long DNA products
-preferential amplification of short products
what does star activity mean?
when restriction enzyme cuts the DNA too many times and there are extra bands
what is the typical dNTP concentration?
0.1-0.5 mM
what happens if you have too much magnesium?
-promotes misincorporation
- more non-specific products
what happens if the annealing temp is too high?
-temp may be too high to allow primer binding
- reduced to no product yield
where can DNA be isolated from in a blood sample?
whole blood or buffy coat
Define: the binding of a single-stranded molecule of DNA to a complementary single stranded target DNA molecule
hybridization
what happens if you have too little magnesium?
-lowers enzyme efficiency
-low yield of PCR product
if the amount of salts in a sample increases, the speed of DNA migration…
decreases
for restriction enzyme mapping, what does the size of the bands indicate?
the distance between the restriction sites?
what is the concentration for taq
0.5-2.5 units/ 50ul reaction
ability to detect only the analyte and not non-specific targets
analytical specificity
List identifiers that could be present on specimens:
Name
Date of birth
Patient ID#
Specimen ID or requisition #
What is the ideal amplicon size for real-time PCR?
50-150bp
Stringency: As time of hybridization increases, strincency
stringency decreases
the long the probe mixes with the DNA, the more likely it will bind non specifically
Define: Taq polymerase adds nucleotides to make new strand
Extension
expected analyte frequency or levels from a population of individuals
reference range
verifying test performance criteria according to the package insert for an FDA approved test can be defined as
verification
List the types of denaturing/destabilizing agents:
Formamide
Urea
TMAC
what is added to a gel to keep RNA denatured?
formaldehyde
if one parent gives you the normal allele and the other parent gives you the mutant allele, what is your genotype?
heterozygous
if you aren’t getting any product should you increase or decrease Mg conc.
increase
the range within which a specimen may be measured directly (w/o dilution or concentration)
analytical measurement range
what is the equation for PPV?
TP
TP+FP
Define: two or more chromosome complements present in a cell
mosaicism
establishing test performance criteria in a lab developed test (LDT, aka “home brew” can be defined as
validation
Stringency: As salt concentration increases, stringency…..
stringency decreases
salt stabilizes DNA which promotes any kind of binding, even non-specific kinds– DNA clumps together
Define: 3 copies of a chromosome instead of 2
trisomy
The presence of what will increase stringency because it promotes denaturation at even lower temperatures?
formamide
If gel concentration is increased, the speed of DNA migration
decreases
mutation R506Q in the coagulation factor V gene that causes a hypercoagulable (thrombophillic) phenotype
Factor V leiden
if buffer volume increases, the speed of DNA migration….
decreases
Stringency: As the time of washing the blot increases, stringency
stringency increases
the longer the wash time, the more likely it will detach weak non-specific bonds, leaving only specific probe-target complexes
agreement between independent test results
precision
if gel size increases, the speed of DNA migration…..
decreases
Probe Tm should be?
10 degrees celsius higher than the primer
this uses DNA immobilized on a solid support. beads or columns
solid phase DNA isolation
define: having only one copy of an X-chromosome
hemizygous
about how many bases long should probes be?
20-30
what is the typical DNA concentration for a PCR reaction?
1ng-100ng of DNA
non-methylated template CG becomes….
UG
what is the ideal magnesium concentration for PCR?
1-4mM
quantitative correlation between test result and actual amount of analyte
linearity
a higher % of gels can separate what kind of fragments?
smaller
Define: diseases with genetic components; not necessarily hereditary; present at birth
congenital
change in assay response with corresponding change in analyte
analytical sensitivity/limit of detection
what are things that can affect PCR efficiency?
primer design, reaction conditions, PCR inhibitors, sample quality.
Define: Dried blood on thick filter paper; can be used for isolation of DNA in newborn screening, future genetic testing
Guthrie Cards
Define: the combination of conditions under which the target is exposed to the probe
stringency
Define: more than two of any autosome
polyploidy
at what concentrations are agarose gels used at?
0.5-5%
Define: oligonucleotide primers hybridize to target DNA
annealing
the adjustment of an instrument or assay result to the actual concentration of a known reference analyte by testing and making the appropriate adjustments
calibration
autosomal recessive condition that causes over absorption of iron from food
hemochromatosis
define: gain or loss of an autosome
aneuploid
what is the optimum A 260/280 ratio for RNA
2.0
(1.7-2.3 is acceptable)
Define: double stranded DNA separated into single strands
denaturation
(Annealing)
if there are non-specific products you should….
increase annealing temp
what are the 3 types of primers used in PCR reactions?
XXXXXX
if both parents give you the same version of a gene, then what is your genotype?
homozygous wildtype
what happens if the annealing temp is too low?
-one or both primers may anneal to sequences other than the target
- non-specific amplification; reduced yield of product
caused by a mutation 677 C>T and 1298 A>C are associated with deficiencies in folate metabolism
MTHFR
Define: containing 3 homologous sets of chromosomes ex: klinefelters
triploid
what is the optimum A260/280 ratio for DNA?
1.8
(1.6-2.1) is acceptable
if the amount of ions in buffer increases, the speed of DNA migration….
increases
What is the equation for clinical sensitivity?
TP
TP+FN
(Annealing)
if there are non-specific products you should……. 2
shorten annealing time
what are the 3 stages of a typical PCR reaction?
baseline range, linear/dynamic range, plateu
Verification of what is required for FDA approval
clinical sensitivity
clinical specificity
accuracy
precision
external specimens supplied by a third party from a known reference source
assesses the competency of the laboratory, blind samples
proficiency testing
methylated template CG remains
CG
range within which test results are considered to be valid (w or w/o dilution)
reportable range
these are probes, primers, antibodies or other test components that detect a specific target
analyte specific reagents
Pyrosequencing is quantitative while Sanger sequencing is……
semi-quantitative
List the reagents of a restriction enzyme master mix:
- 1X RE buffer
- Restriction enzyme
- stabilizers (BSA, spermidine)
- water
- DNA sample
total volume should be 25-50uL
(Annealing)
if there is no product you should
you should decrease annealing temp
this uses organic chemicals, phenol, and cholorform
organic DNA isolation
caused by loss of function of the CFTR gene; codes for a chloride channel membrane protein
cystic fibrosis
Stringency: As temperature increases, stringency……
stringency increases
ability of test results to predict a clinical condition
clinical sensitivity
where are restriction endonucleases found in nature?
bacterial cells
for restriction enzyme mapping, what does the number of bands indicate?
the number of restriction sites
disease associated results only in patients who actually have the disease conditions
clinical specificity
List the steps of southern blotting:
- RE digest
- Electrophoresis of an agarose gel
- transfer DNA to a nitrocellulose membrane (blotting)
- hybridize probe to blot (wash to remove unbound probe)
- detection of probe signal
if gel thickness increases, the speed of DNA migration….
decreases
Define: one copy of a chromosome instead of 2
monosomy
If dyes are added to gel, the speed of DNA migration…..
decreases
a mutation in the 3’ UTR of the gene that codes for prothrombin or coagulation factor II; results in an autosomal dominant increased risk for thrombosis
Prothrombin
what is the equation for clinical specificity?
TN
TN+FP
what is the typical final concentration for each primer?
100nM to 1uM
if you need high fidelity do you want increase or decrease Mg conc?
decrease
if you increase the temperature, the speed of DNA migration
increases
Stringency: As denaturing/destabilizng agents increase, stringency…..
stringency increases
When probe and target are prone to denaturation, only the most specific probe target hybridization can occur
what kind of appearance will partially degraded RNA have?
smeared
what are the concentrations that polyacrylamide gels are used at?
3.5-20%
which is better for separating larger DNA fragments? agarose or polyacrylamide gels?
agarose
A lower % of gels can separate what kind of fragments?
larger
if both parents give you the “mutant” or “minor” allele, what is your genotype?
homozygous mutant
primers and probes designed for real time PCR should have a GC content of?
30-80%
about how many bases long should primers be?
20-24 bases long
RNA gels can be stained with:
ethidium bromide, acridine orange, SYBR green II
when isolating RNA using the TRIzol procedure, where is RNA found?
aqueous phase
When isolating RNA using the TRIzol procedure, where is the DNA found?
interphase
when isolating RNA using the TRIzol procedure, what can be found in the organic phase?
proteins and lipids
If you are looking to isolate mRNA specifically, what else would you need to do?
Bind the poly A tail with a poly T resin or bead
List the types of positive controls:
-general positive controls
- amplification/inhibition controls
- internal controls
If a positive control fails, is the run valid?
NO! Positive controls should always be positive, if not then the assay is valid
Amplification control must be (positive/negative) in assay target NEGATIVE specimens, otherwise it could be a false negative
positive
What do amplification controls rule out?
false negative
what does heterologous means when it comes to primers?
internal control primers amplify a different sequences than the assay target
This type of internal control sequence is added to the reaction
extrinsic
this type of internal control sequence is already present in the sample
intrinsic
what is it called when two different products are produced in the same reaction?
multiplexed
this type of control is also known as the housekeeping gene
heterologous intrinsic controls
what are the two major types of negative controls?
reagent blank/contamination control
-negative template control
this type of control is always negative, contains all reagents except target sequences, and checks for false positive results
reagent blank/contamination control
this type of control should always be negative, and it does not contain the target sequence but may contain other sequences.
negative template control
Do you have to have a different set of primers for your intrinsic and extrinsic controls?
yes
If the positive control fails, the run is
invalid
if the amplification/internal control fails for a sample, the run
only that individual sample is considered a failure, only the failed sample needs to be repeated
if the negative control fails, the run is
invalid
