Term
| DNA target, 2 primers, Taq DNA polymerase, deoxynucleotide, and buffer of MG are all components of ___? |
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Definition
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Term
| How many primers are needed for each strand? |
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Definition
1 per strand
1 complimentary to one strand
and 1 complimentary to the other strand |
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Term
| What does thermophilic mean? |
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Definition
|
|
Term
| What is the source of Taq DNA polymerase? |
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Definition
|
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Term
| What is Taq DNA polymerase optimal growth temp? |
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Definition
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Term
| How much activity does Taq have at 37 degrees C? |
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Definition
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Term
| What temp does Taq become inactive? |
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Definition
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Term
| Denaturation, Annealing,and Elongation are all steps for? |
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Definition
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Term
| What temp is used in the denaturation step? |
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Definition
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Term
| How does the heat in the deanturation stage separate the strands? |
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Definition
| It breaks the hydrogen bonds |
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Term
| What temps are used in the annealing stage? |
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Definition
| temp is reduced to 55 to 65 degrees C |
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Term
| What do the primers anneal to in the annealing stage? |
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Definition
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Term
| What temp is used in the elongation step? |
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Definition
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Term
| What step does Taq DNA polymerase synthesizes new complementary DNA strands? |
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Definition
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Term
| HOw many times are cycles repeated? |
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Definition
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Term
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Definition
|
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Term
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Definition
|
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Term
| Denaturation + Annealing + Extensions steps = ??? |
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Definition
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Term
| How does more G-C in the annealing step affect the temperature? |
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Definition
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Term
|
Definition
| (pcr product) detection/ analysis |
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Term
| PCR efficiency is affected by? |
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Definition
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Term
| Standard PCR can ammplify up to?? |
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Definition
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Term
| With modifications PCR can amplify up to? |
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Definition
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Term
| Taq DNA polymerase, dNTPs, primers and buffer are all in the?? |
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Definition
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Term
| T OR F? For QA there are 3 distinct work areas (reagent area, extraction area, PCR area) and the work flow is unidirectional? |
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Definition
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Term
Is this an example of work flow unidirectional?
Reagant--> Extractions --> PCR |
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Definition
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Term
| Name some other procedures for reducing contamination? |
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Definition
Store reagents in small aliquots, discard after one use
use aerosol resistant tips for pipettes
use disposable plastic ware
clean areas with bleach
avoid splashing and aerosols
open only one specimen tube at a time
Keep tops on reagents
pulse spin tubes before opening |
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Term
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Definition
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Term
| Why should you pulse spin tubes before opening? |
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Definition
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Term
| Patient samples are processed in which work area? |
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Definition
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Term
| Thermal cyclers and analytical instruments are used in which area? |
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Definition
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Term
| What transcribes RNA into DNA? |
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Definition
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Term
| What is the purpose of reverse transcriptase? |
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Definition
to detect RNA in patient specimens
such as: detect RNA viruses (HIV, HCV) and to determine gene "expression" |
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Term
| __________ = Complementary DNA (cDNA) |
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Definition
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Term
| mRNA has what type of tail? |
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Definition
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Term
| What primer is needed in reverse transcriptase? |
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Definition
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Term
| What differs from genomic and cDNA? |
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Definition
Genomic DNA: introns and exons
cDNA is a copy of mRNA (no introns) |
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Term
| Are viral primers specific or nonspecific? |
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Definition
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Term
| What is the amplification of 2 or more target sequences in one rxn? |
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Definition
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Term
| In multiplex PCR a primer is needed for each? |
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Definition
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Term
| What is the purpose of Multiplex PCR? |
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Definition
| ID species and at the same time detect target sequences |
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Term
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Definition
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Term
| When and how are amplicons detected in Traditional PCR (End point PCR) |
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Definition
amplicon detected at the end of series of rxns
semi-quantitative
distinguishes amplicons based on size
not automated
requires post PCR processing |
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Term
| What is an example of post PCR processing? |
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Definition
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Term
| When and how are amplicons detected in real time PCR? |
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Definition
uses chemicals to detect PCR amplification during early phase of RXNS
can quantitate starting amount of nucleic acid |
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Term
| Exponential, Linear and Plateau are ? |
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Definition
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Term
Reaction specific and precise
Exact doubling of product every cycle
phase used in real time PCR
These are all apart of which PCR phase? |
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Definition
| Exponential (aka geometric) |
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Term
High variability
Rxn components being consumed
rxn slowing
products start to degrade
These are all apart of which PCR phase? |
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Definition
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|
Term
End point for gel detection
Rxn has stopped
no more products are being made
PCR products will degrade if left too long
These are all apart of which PCR phase? |
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Definition
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|
Term
| What is the Ct = cycle threshold? |
|
Definition
| number of PCR cycles to reach threshold where fluorescence is detected |
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|
Term
| What does a high conc mean for Ct? |
|
Definition
| will need fewer cycles to reach Ct |
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Term
| What does a low conc mean for Ct? |
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Definition
| will need more cycles to reach Ct |
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Term
What probe does the TaqMan method use?
Hydrolysis or Hybridization? |
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Definition
|
|
Term
| What are the fluorophores used in the TaqMan Method? |
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Definition
|
|
Term
| What does FRET stand for? |
|
Definition
| Fluorescence Resonance Energy Transfer |
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Term
|
Definition
| one dye (Q) interferes with fluorescence of the other (R) |
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|
Term
Principle/Procedure for?
Target DNA denatured to single stands
Primer binds to template/target DNA
TaqMan probe binds to template/target DNA
Taq polymerase synthesizes new DNA. (when it reaches TaqMan probe, Reporter is removed)
Reporter is now free of Q and can fluoresce |
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Definition
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Term
| What probe does the Molecular Beacon Method use? |
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Definition
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|
Term
| Molecular Beacon Method is ____ based technology |
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Definition
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|
Term
Principle/Process for?
PCR portion of this test is standard
Primers included in Master Mix
When newly synthesized PCR products denatured by heat- beacon molecules "ends" also denature and the target specific nucleotides are now available to attach to specific target during annealing phase
When beacon hybridizes to target.
-R and Q sep by distance
-R can now fluoresce
-R and Q still attached to nucleotide chain |
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Definition
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Term
| When the Beacon forms a hairpin the ends are ? |
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Definition
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|
Term
Which method is this?
Fluorescent probe
SYBR green probe binds to double stranded DNA
Non-specific binding; can combine to any double stranded DNA
Fluorescence increases as PCR increases amt of DNA |
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Definition
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Term
Which are target, signal or probe amplification tests?
Hybrid capture assay?
Branched chain DNA (bDNA) assay?
Cleavase Invader technology? |
|
Definition
Signal: hybrid capture and bDNA
Probe: cleavase invader |
|
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Term
Summary of?
Denature target DNA
SS-DNA + RNA probe --> RNA-DNA hybrid
RNA-DNA hybrid binds to anti-hybrid abys attached to tube
2nd aby (w enzyme) added, aby binds to hybrid attached to tube
add substrate, enzyme + substrate --> light
measure amount of light |
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Definition
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Term
| The amount of light is _____ to amount of target DNA |
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Definition
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Term
Summary of??
Target+ capture probe --> target attached to tube
attached target then layered with several probe types
top layer probe has enzyme label
add enzyme substrate --> light
measure amount of light |
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Definition
| Branched chain DNA (bDNA) assay |
|
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Term
| Cleavase invader technology uses two oligonucleotide probes? |
|
Definition
Primary probe- specific for target
invader oligo |
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Term
| The part of the primary probe that does not bind to the target is? |
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Definition
|
|
Term
| cleavase removes the flap --> |
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Definition
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|
Term
| The primary rxn of the cleavase invader tech includes two things? |
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Definition
| the flap and the cleaved flap |
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Term
| In the ___ rxn the cleaved flap attaches to fret casette which forms another flap. cleavase removes the casette flap and releases reporter. which now fluoresces |
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Definition
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Term
| Amplification occurs in two places in the cleavase invader tech. where? |
|
Definition
| attachment of primary and invader probes to target cycles and cleaved flap |
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Term
| Cleavase invader tech can detect what type of mutations |
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Definition
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|
Term
| Cleavase invader is not as versatile as?? |
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Definition
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|
Term
| What percent of the human genome is the same? |
|
Definition
|
|
Term
| What percent difference is used to distinguish individuals |
|
Definition
|
|
Term
| Restriction Fragment Length Polymorphisms (RFLP) was discovered when by who? |
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Definition
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|
Term
| The number of repeats at any particular location differs from person to person this is known as? |
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Definition
| VNTR- variable number tandem repeats |
|
|
Term
| What test is used to analyze VNTRs? |
|
Definition
|
|
Term
| What describes an individuals unique banding pattern? |
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
| they are only 1-8 nucleotides long |
|
|
Term
| Are VNTRs or STRs inherited from the parents? |
|
Definition
|
|
Term
| Minisatellites and minisatellite DNA are synonyms for ? |
|
Definition
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|
Term
| Microsatellites and microsatellite DNA are synonyms for? |
|
Definition
|
|
Term
|
Definition
|
|
Term
| What occurs in multiple genes? |
|
Definition
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|
Term
| Do VNTRs or STRs have noncoding regions? |
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Definition
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|
Term
| Being difficult to analyze, requires a moderate amount of intact DNA and often takes several days to perform are disadvantages of what? |
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Definition
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|
Term
| PCR can be used bc DNA is shorter, requires small amounts of DNA and requires fewer copies of intact DNA in a degraded specimen and can be automated making it able to be performed in several hours are advantages of? |
|
Definition
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Term
| A federally maintained database of DNA obtained from crime scenes or convicted violent offenders is called? |
|
Definition
| CODIS- Combined DNA Index System |
|
|
Term
| What is the number and type of loci present in CODIS? |
|
Definition
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|
Term
| 13 STR loci is amplified by what? |
|
Definition
|
|
Term
| What does the amelogenin locus determine? |
|
Definition
|
|
Term
| p^2 (AA) = 2pq (Aa) = q^2 (aa) = 1 |
|
Definition
RMP calculation
Random Match Probability |
|
|
Term
| What is the locus frequency calculation for heterozygous and homozygous? |
|
Definition
Hetero: 2pq
Homo: p^2 or q^2 |
|
|
Term
Always wear gloves
Tie hair back or cover it
Sneezing or coughing expels cells
and be careful not accidentially transfer biologics
These are all apart of? |
|
Definition
| Crime scene specimen collection |
|
|
Term
| What is the most important step in crime scene specimen collections? |
|
Definition
| improper collection may invalidate test results |
|
|
Term
| What do you get from maternal inheritance only? |
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Definition
|
|
Term
| WHat is non-nuclear, double stranded DNA? |
|
Definition
|
|
Term
| What cycle gives mtDNA genes their energy? |
|
Definition
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|
Term
| What is more stable than DNA and has a high copy number and has hypervariable region between individuals? |
|
Definition
|
|
Term
| What is mtDNA useful for? |
|
Definition
| identifying individuals of disaster and for cold cases |
|
|
Term
|
Definition
| Amplified fragment length polymorphisms |
|
|
Term
| Do AFLP have specific sequences amplified? |
|
Definition
|
|
Term
|
Definition
| Single nucleotide polymorphisms |
|
|
Term
| Do SNPs have single base pair polymorphisms |
|
Definition
|
|
Term
|
Definition
| silent single nucleotide polymorphisms |
|
|
Term
| Do SSNPs affect gene product or regulation |
|
Definition
|
|
Term
| What has many markers (10 million) and is more costly than STR analysis |
|
Definition
|
|
Term
| Where is a Y chromosome inherited from |
|
Definition
|
|
Term
| The Y chromosome is found only in |
|
Definition
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