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What are the 5 components of PCR?
-Deoxynucleotide triphosphates (A, C, T, G)
Short synthetic oligonucleotides consisting of 20 to 30 bases in length that start the PCR reaction
What temperature is the denaturation step in which the DNA is separated into individual strands?
94 degrees Celsius
What temperature range is the annealing phase in which primers anneal to denatured DNA strands?
55 to 65 degrees Celsius
What temperature is the elongation step in which Taq polymerase synthesizes new complementary DNA strands?
72 degrees Celsius
How many sets of Precise Length DNA are present after 20 cycles?
1,048,576 (2 to the 20th exponential power)
To decrease the possibility of DNA cross-contamination, workflow in a molecular lab is ______________?
What is the order of the workstations in a PCR lab?
Reagent station --> Extraction hood --> PCR (Thermal Cycler & Electrophoresis machines)
What is the purpose of the reagent station in a PCR lab?
To prepare the master mix (primers and Taq polymerase)
To avoid contamination, how should reagents be handled in the PCR lab?
Aliquot large amounts of reagent into several small containers and use the aliquot container only once (once the container is open it must be used or disposed of; do not put it back in storage)
What cleaner is used to clean any surface that has been used to prepare a PCR sample?
Bleach (it destroys DNA)
How often should you clean surfaces used to prepare a PCR sample?
Before, during and after every sample
Is it okay to prepare your master mix in glassware that has been thoroughly cleansed and bleached?
No; use disposable plasticware only
What unique centrifugation method is used in the PCR lab to prevent contamination?
Pulse spin; it reduces the possibility of the sample becominig aerosolized in the centrifuge
To avoid contamination, what types of tubes are used in a PCR lab?
Tubes with a screwcap top are used to avoid accidental splashing
DNA polymerase enzyme that transcribes single-stranded RNA into single-stranded DNA
What is the purpose of reverse transcriptase PCR?
To convert retrovirus (Hepatitis C & HIV) RNA into DNA that can be amplified using PCR to detect the virus and to measure gene expression
Genomic DNA has both introns and exons. Does cDNA also have introns and exons?
cDNA only has exons; cDNA is synthesized from mRNA, therefore there are no introns
What primer is necessary for the reverse transcriptase enzyme to transcribe RNA into cDNA for the purpose of measuring gene expression? Why is it necessary?
poly(T) oligonucleotide primer; poly(T) binds to the poly(A) tail of mRNA and reverse transcriptase binds to the poly(T)
What primer is necessary for the reverse transcriptase enzyme to transcribe RNA into cDNA for the purpose of viral identification?
Sequence specific primers
How many targets and primers are necessary for multiplex PCR?
Need a 2-primer set for each DNA target
What happens during the Exponential phase of PCR amplification?
Exact doubling of product every cycle
What happens during the Linear phase of PCR amplification?
Reaction components are being consumed, leading to high variability in amplicon production
What happens during the Plateau phase of PCR amplification?
Components are completely consumed, leading to no more products being made
Fluorescence Resonance Energy Transfer
One dye (quencher) interferes with fluorescence of the other (reporter)
How does the reporter dye fluoresce in the TaqMan method?
The TaqMan probe (reporter dye and quencher dye) binds to the template DNA. As Taq polymerase is synthesizing DNA, when it reaches the TaqMan probe it removes the reporter and it can now fluoresce
How does the reporter dye fluoresce in the Molecular Beacon method?
As newly synthesized PCR products are denatured by heat, the beacon molecule "ends" also denature. Target specific nucleotides in beacon molecule attach to specific target during annealing phase. When beacon hybridizes the target, the reporter and quencher are separated by distance and the reporter dye can now fluoresce.
What is unique about the SYBR Green fluorescence method?
Non-specific; probe will bind to any double-stranded DNA; fluorescence increases as PCR increases amount of ds-DNA
When you take a fluorescent signal and multiply it repeatedly amplifying the strength of the signal rather than the target
In signal amplification, what is the number of target sequences
The number of target sequences stays the same (the target is not amplified... the fluorescent signal is)
A DNA:RNA hybrid is bound to the microtiter well by an antibody. Then a 2nd antibody is added, along with an enzyme and substrate. The combination of the enzyme and substrate generates a fluorescent signal and the amount of light is measured.
Hybrid-Capture Signal Amplification
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
Branched-Chain DNA (bDNA) Signal Amplification
What are the components of Branched-Chain DNA signal amplification?
What are the components of Cleavase-Invader probe amplification?
Which components of Cleavase-Invader probe amplification are involved in the primary reaction?
-Primary probe specific for target
If the primary probe and the invader probe are a match, the primary probe will bind to the target DNA
Primary Cleavase reaction
Which enzyme clips the "flap" on the primary probe to bind to the FRET cassette in Cleavase-Invader probe amplification?
What reaction stage is the fluorescent signal generated in Cleavase-Invader probe amplification?
Secondary Cleavase reaction
When light is produced in signal and probe amplification methods, is the light proportional or inversely proportional to the amount of target DNA in the sample?
Combined DNA Index System; a federally maintained database of DNA obtained from crime scenes and convicted violent offenders
What crime scene specimen collection procedures must be followed?
-Always wear gloves
-Tie hair back or cover it
-Do not cough or sneeze
-If collecting multiple samples, take care to minimize cross-contamination
What is the formula to calculate the random match probability?
f(locus 1) X f(locus 2) X f(locus 3) = RMP
What are mitochondrial DNA fingerprinting techniques used for?
-Identifying disaster victims
Testing procedure based on the amplification of DNA. A variation of RFLP where specific sequences are amplified.
Amplified Fragment Length Polymorphism (AFLP)
Single base pair polymorphism that doesn't affect gene production or gene regulation
Silent Single Nucleotide Polymorphism (SSNP)
Who approved the guidelines that scientists at the Asilomar Conference in California set for genetic engineering?
Who won the race between Genentech and Walter Gilbert? How?
Genentech; they manufactured synthetic DNA
Which DNA profiling technique was used in the first death sentence case involving DNA fingerprinting? Why?
Single-locus; less ambiguous and precise allele determination
Who was Anna Anderson?
Franziska Schanzkowska, a Polish factory worker with a history of mental illness
True/False: Once critical mutation occurs, cancer evolves by accumulating more mutations in genes controlling cell growth
What are the functions of proto-oncogenes?
Promoting cell proliferation & inhibiting cell death (apoptosis)
What are the functions of tumor suppressor genes?
Regulating cell proliferation & repairing damaged DNA
What is the result of a mutated tumor suppressor gene?
Proliferation no longer down-regulated & apoptosis no longer promoted; cells proliferate uncontrollably
Alters activity of transcription factors & controls cell division; tumor suppressor gene
Rb (Retinal blastoma)
How does a mutation in the BRCA gene cause breast cancer?
Tumor suppressor gene is lost; over time leads to malignancy
Did Mary-Claire King and Mark Skolnick work closely together to find the "breast cancer gene?"
No; they were competing
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