AMT FINAL Flashcards
1
Q
what are the 3 types of dyes used to dye gels?
A
ethidium bromide, SYBR green, gel red
2
Q
A good standard curve must have the following properties:
A
-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
3
Q
what is the ideal GC content when designing primers?
A
50-60%
4
Q
denaturant, lowers denaturing temp
-used with high secondary structure (GC-rich DNA)
A
formamide
5
Q
what happens if you increase salt in a PCR reaction?
A
-slows denaturation of long DNA products
-preferential amplification of short products
6
Q
what does star activity mean?
A
when restriction enzyme cuts the DNA too many times and there are extra bands
7
Q
what is the typical dNTP concentration?
A
0.1-0.5 mM
8
Q
what happens if you have too much magnesium?
A
-promotes misincorporation
- more non-specific products
9
Q
what happens if the annealing temp is too high?
A
-temp may be too high to allow primer binding
- reduced to no product yield
10
Q
where can DNA be isolated from in a blood sample?
A
whole blood or buffy coat
11
Q
Define: the binding of a single-stranded molecule of DNA to a complementary single stranded target DNA molecule
A
hybridization
12
Q
what happens if you have too little magnesium?
A
-lowers enzyme efficiency
-low yield of PCR product
13
Q
if the amount of salts in a sample increases, the speed of DNA migration…
A
decreases
14
Q
for restriction enzyme mapping, what does the size of the bands indicate?
A
the distance between the restriction sites?
15
Q
what is the concentration for taq
A
0.5-2.5 units/ 50ul reaction
16
Q
ability to detect only the analyte and not non-specific targets
A
analytical specificity
17
Q
List identifiers that could be present on specimens:
A
Name
Date of birth
Patient ID#
Specimen ID or requisition #
18
Q
What is the ideal amplicon size for real-time PCR?
A
50-150bp
19
Q
Stringency: As time of hybridization increases, strincency
A
stringency decreases
the long the probe mixes with the DNA, the more likely it will bind non specifically
20
Q
Define: Taq polymerase adds nucleotides to make new strand
A
Extension
21
Q
expected analyte frequency or levels from a population of individuals
A
reference range
22
Q
verifying test performance criteria according to the package insert for an FDA approved test can be defined as
A
verification
23
Q
List the types of denaturing/destabilizing agents:
A
Formamide
Urea
TMAC
24
Q
what is added to a gel to keep RNA denatured?
A
formaldehyde
25
if one parent gives you the normal allele and the other parent gives you the mutant allele, what is your genotype?
heterozygous
26
if you aren't getting any product should you increase or decrease Mg conc.
increase
27
the range within which a specimen may be measured directly (w/o dilution or concentration)
analytical measurement range
28
what is the equation for PPV?
TP
TP+FP
29
Define: two or more chromosome complements present in a cell
mosaicism
30
establishing test performance criteria in a lab developed test (LDT, aka "home brew" can be defined as
validation
31
Stringency: As salt concentration increases, stringency.....
stringency decreases
salt stabilizes DNA which promotes any kind of binding, even non-specific kinds-- DNA clumps together
32
Define: 3 copies of a chromosome instead of 2
trisomy
33
The presence of what will increase stringency because it promotes denaturation at even lower temperatures?
formamide
34
If gel concentration is increased, the speed of DNA migration
decreases
35
mutation R506Q in the coagulation factor V gene that causes a hypercoagulable (thrombophillic) phenotype
Factor V leiden
36
if buffer volume increases, the speed of DNA migration....
decreases
37
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
38
agreement between independent test results
precision
39
if gel size increases, the speed of DNA migration.....
decreases
40
Probe Tm should be?
10 degrees celsius higher than the primer
41
this uses DNA immobilized on a solid support. beads or columns
solid phase DNA isolation
42
define: having only one copy of an X-chromosome
hemizygous
43
about how many bases long should probes be?
20-30
44
what is the typical DNA concentration for a PCR reaction?
1ng-100ng of DNA
45
non-methylated template CG becomes....
UG
46
what is the ideal magnesium concentration for PCR?
1-4mM
47
quantitative correlation between test result and actual amount of analyte
linearity
48
a higher % of gels can separate what kind of fragments?
smaller
49
Define: diseases with genetic components; not necessarily hereditary; present at birth
congenital
50
change in assay response with corresponding change in analyte
analytical sensitivity/limit of detection
51
what are things that can affect PCR efficiency?
primer design, reaction conditions, PCR inhibitors, sample quality.
52
Define: Dried blood on thick filter paper; can be used for isolation of DNA in newborn screening, future genetic testing
Guthrie Cards
53
Define: the combination of conditions under which the target is exposed to the probe
stringency
54
Define: more than two of any autosome
polyploidy
55
at what concentrations are agarose gels used at?
0.5-5%
56
Define: oligonucleotide primers hybridize to target DNA
annealing
57
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
58
autosomal recessive condition that causes over absorption of iron from food
hemochromatosis
59
define: gain or loss of an autosome
aneuploid
60
what is the optimum A 260/280 ratio for RNA
2.0
(1.7-2.3 is acceptable)
61
Define: double stranded DNA separated into single strands
denaturation
62
(Annealing)
if there are non-specific products you should....
increase annealing temp
63
what are the 3 types of primers used in PCR reactions?
XXXXXX
64
if both parents give you the same version of a gene, then what is your genotype?
homozygous wildtype
65
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
66
caused by a mutation 677 C>T and 1298 A>C are associated with deficiencies in folate metabolism
MTHFR
67
Define: containing 3 homologous sets of chromosomes ex: klinefelters
triploid
68
what is the optimum A260/280 ratio for DNA?
1.8
(1.6-2.1) is acceptable
69
if the amount of ions in buffer increases, the speed of DNA migration....
increases
70
What is the equation for clinical sensitivity?
TP
TP+FN
71
(Annealing)
if there are non-specific products you should....... 2
shorten annealing time
72
what are the 3 stages of a typical PCR reaction?
baseline range, linear/dynamic range, plateu
73
Verification of what is required for FDA approval
clinical sensitivity
clinical specificity
accuracy
precision
74
external specimens supplied by a third party from a known reference source
assesses the competency of the laboratory, blind samples
proficiency testing
75
methylated template CG remains
CG
76
range within which test results are considered to be valid (w or w/o dilution)
reportable range
77
these are probes, primers, antibodies or other test components that detect a specific target
analyte specific reagents
78
Pyrosequencing is quantitative while Sanger sequencing is......
semi-quantitative
79
List the reagents of a restriction enzyme master mix:
1. 1X RE buffer
2. Restriction enzyme
3. stabilizers (BSA, spermidine)
4. water
5. DNA sample
total volume should be 25-50uL
80
(Annealing)
if there is no product you should
you should decrease annealing temp
81
this uses organic chemicals, phenol, and cholorform
organic DNA isolation
82
caused by loss of function of the CFTR gene; codes for a chloride channel membrane protein
cystic fibrosis
83
Stringency: As temperature increases, stringency......
stringency increases
84
ability of test results to predict a clinical condition
clinical sensitivity
85
where are restriction endonucleases found in nature?
bacterial cells
86
for restriction enzyme mapping, what does the number of bands indicate?
the number of restriction sites
87
88
disease associated results only in patients who actually have the disease conditions
clinical specificity
89
List the steps of southern blotting:
1. RE digest
2. Electrophoresis of an agarose gel
3. transfer DNA to a nitrocellulose membrane (blotting)
4. hybridize probe to blot (wash to remove unbound probe)
5. detection of probe signal
90
if gel thickness increases, the speed of DNA migration....
decreases
91
Define: one copy of a chromosome instead of 2
monosomy
92
If dyes are added to gel, the speed of DNA migration.....
decreases
93
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
94
what is the equation for clinical specificity?
TN
TN+FP
95
what is the typical final concentration for each primer?
100nM to 1uM
96
if you need high fidelity do you want increase or decrease Mg conc?
decrease
97
if you increase the temperature, the speed of DNA migration
increases
98
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
99
what kind of appearance will partially degraded RNA have?
smeared
100
what are the concentrations that polyacrylamide gels are used at?
3.5-20%
101
which is better for separating larger DNA fragments? agarose or polyacrylamide gels?
agarose
102
A lower % of gels can separate what kind of fragments?
larger
103
if both parents give you the "mutant" or "minor" allele, what is your genotype?
homozygous mutant
104
primers and probes designed for real time PCR should have a GC content of?
30-80%
105
about how many bases long should primers be?
20-24 bases long
106
RNA gels can be stained with:
ethidium bromide, acridine orange, SYBR green II
107
when isolating RNA using the TRIzol procedure, where is RNA found?
aqueous phase
108
When isolating RNA using the TRIzol procedure, where is the DNA found?
interphase
109
when isolating RNA using the TRIzol procedure, what can be found in the organic phase?
proteins and lipids
110
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
111
List the types of positive controls:
-general positive controls
- amplification/inhibition controls
- internal controls
112
If a positive control fails, is the run valid?
NO! Positive controls should always be positive, if not then the assay is valid
113
Amplification control must be (positive/negative) in assay target NEGATIVE specimens, otherwise it could be a false negative
positive
114
What do amplification controls rule out?
false negative
115
what does heterologous means when it comes to primers?
internal control primers amplify a different sequences than the assay target
116
This type of internal control sequence is added to the reaction
extrinsic
117
this type of internal control sequence is already present in the sample
intrinsic
118
what is it called when two different products are produced in the same reaction?
multiplexed
119
this type of control is also known as the housekeeping gene
heterologous intrinsic controls
120
what are the two major types of negative controls?
reagent blank/contamination control
-negative template control
121
this type of control is always negative, contains all reagents except target sequences, and checks for false positive results
reagent blank/contamination control
122
this type of control should always be negative, and it does not contain the target sequence but may contain other sequences.
negative template control
123
Do you have to have a different set of primers for your intrinsic and extrinsic controls?
yes
124
If the positive control fails, the run is
invalid
125
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
126
if the negative control fails, the run is
invalid
127
