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Includes material from exams 3 & 4

What are the 5 components of PCR?

-Taq Polymerase
-DNA Target
-Deoxynucleotide triphosphates (A, C, T, G)

Short synthetic oligonucleotides consisting of 20 to 30 bases in length that start the PCR reaction


How many primers are needed for a single target?

2 (one for each strand of DNA)

How many primers are needed for multiple targets?

2 for each target

What organism is Taq polymerase isolated from?

Thermus aquaticus bacteria

What is the optimal temperature for Taq polymerase to activate?

70 degrees Celsius

What chemical component is found in PCR buffer?


What is the DNA target?

The DNA strand to be amplified

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

The target sequence _______ with each cycle.


How many cycles does it take to perform PCR?

20 to 40

How much time does it take for one temperature cycle?

5 minutes

The original DNA strand to be amplified

Template DNA

How many sets of Template DNA are present after 20 cycles?


How many sets of Intermediate DNA are present after 20 cycles?

20 (1 set per cycle)

How many sets of Precise Length DNA are present after 20 cycles?

1,048,576 (2 to the 20th exponential power)

DNA that is a product of PCR


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)

Are patient samples authorized in the reagent station of a PCR lab?


What is the purpose of the reagent station in a PCR lab?

To prepare the master mix (primers and Taq polymerase)

To avoid contamination you should only open ___ specimen tube at a time


What type of micropipette tips are allowed in the PCR lab?

Aerosol Resistant Tips (ART tips)

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

Reverse transcriptase

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

DNA synthesized from RNA by reverse transcriptase enzyme

Complementary DNA (cDNA)

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

Amplification of 2 or more target sequences in one reaction?

Multiplex PCR

How many targets and primers are necessary for multiplex PCR?

Need a 2-primer set for each DNA target

What is the purpose of multiplex PCR?

To ID species & detect target sequence simultaneously

In end-point PCR, the amplicon is detected _____ of the reaction?

At the end

In real-time PCR, the amplicon is detected _______ of the reaction?

During the early phase

End-point PCR is _____________?


Real-time PCR is ___________?


What are the 3 phases of PCR amplification?

Exponential, Linear & Plateau

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

In what phase of PCR amplification is the reaction precise & specific


The number of PCR cycles to reach threshold where fluorescence is detected

Cycle threshold (Ct)

What does a low Ct number mean?

High initial concentration; needs fewer cycles to reach Ct

What does a high Ct number mean?

Low initial concentration; needs more cycles to reach Ct

Fluorescence Resonance Energy Transfer

One dye (quencher) interferes with fluorescence of the other (reporter)

What tests use FRET technology?

-Molecular Beacon
-Cleavase Invader

Which fluorescence method uses a hydrolysis probe?


Which fluorescence method uses a hybridization probe?

Molecular Beacon

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.

Which tests are real-time PCR tests?

-Molecular Beacon
-SYBR Green

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

Which tests are signal amplification tests?

Hybrid-Capture & branched-chain DNA (bDNA)

Which test is a probe amplification test

Cleavase invader

In target amplification, you start with one piece of DNA and make ________ of copies


When you take a fluorescent signal and multiply it repeatedly amplifying the strength of the signal rather than the target

Signal Amplification

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

Which amplification method is a solid-phase or "sandwich" hybridization?

Branched-Chain DNA (bDNA)

What are the components of Branched-Chain DNA signal amplification?

-Extender Probe
-Pre-amplifier Probe
-Amplifier Probe

What are the components of Cleavase-Invader probe amplification?

-Target DNA
-Primary probe
-Invader oligo
-FRET cassette
-Cleavase enzyme

How many reactions are involved in Cleavase-Invader probe amplification?

2; primary and secondary

Which components of Cleavase-Invader probe amplification are involved in the primary reaction?

-Target DNA
-Primary probe specific for target
-Invader oligonucleotide

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?


What percentage of DNA is different between humans to distinguish individuals?


DNA sequences that repeat

Variable Number Tandem Repeats (VNTR)

Short DNA sequences that repeat

Short Tandem Repeat (STR)

How long is a VNTR?

8 to 90 base pairs in length

How long is a STR?

1 to 8 base pairs in length

Are VNTRs in the coding or non-coding regions?


Are STRs in the coding or non-coding regions?


Which DNA fingerprinting technique was discovered first?

Variable Number Tandem Repeats (VNTR)

What progenitor does a person get their VNTRs?

From both parents

What progenitor does a person get their STRs?

From both parents

How are VNTRs analyzed?

Restriction Fragment Length Polymorphisms (Southern Blot)

How are STRs analyzed?


What quality of DNA is acceptable for VNTR analysis?

Good DNA

What quality of DNA is acceptable for STR analysis?

Degraded DNA may be used

What is a synonym for Variable Number Tandem Repeats (VNTRs)?

Mini-Satellite DNA

What is a synonym for Short Tandem Repeats (STRs)?

Micro-Satellite DNA

A repeat that occurs only once in the genome

Single Locus

A repeat that occurs in multiple places througout the genome


Which portion of the genome (intron/exon) are the VNTRs/STRs located?


Combined DNA Index System; a federally maintained database of DNA obtained from crime scenes and convicted violent offenders


How many loci are analyzed for the CODIS database?

13 STRs and amelogenin locus

What is the purpose of analyzing the amelogenin locus (X/Y Chromosome)?

Determines gender

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 formula is used to calculate a homozygous locus frequency?

p^2 or q^2

What formula is used to calculate a heterozygous locus frequency?


What is the formula to calculate the random match probability?

f(locus 1) X f(locus 2) X f(locus 3) = RMP

Non-nuclear, double-stranded DNA inherited only from your mother

Mitochondrial DNA

What shape is mitochondrial DNA?


Which is more stable? Mitochondrial DNA or chromosomal DNA?

Mitochondrial DNA

Which type of DNA has a hypervariable region?

Mitochondrial DNA

Which type of DNA has a high copy number?

Mitochondrial DNA

What are mitochondrial DNA fingerprinting techniques used for?

-Cold cases
-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

Single Nucleotide Polymorphism (SNP)

Single base pair polymorphism that doesn't affect gene production or gene regulation

Silent Single Nucleotide Polymorphism (SSNP)

Y chromosome is only found in which gender?


Y chromosomes are inherited from which parent?


Power of Discrimination and speed of analysis:
RFLP Analysis

-High discrimination
-Slow speed

Power of Discrimination and speed of analysis:

-Low discrimination
-Slow speed

Power of Discrimination and speed of analysis:
STR analysis

-High discrimination
-Fast speed

Power of Discrimination and speed of analysis:
Blood group typing

-Low discrimination
-Fast speed

Can DNA be transferred from one species to another?


Is DNA transferred from one species to another functional?


Did scientists at the Asilomar Conference in California set guidelines for genetic engineering?


Who approved the guidelines that scientists at the Asilomar Conference in California set for genetic engineering?

Federal government

Does James Watson advocate tight controls on genetic engineering?


In the United States, can living things be patented?


What was the function of the living organism that was first to be patented?

To eat oil

Genentech and Walter Gilbert were in a race to find a gene that codes for what protein?


Who won the race between Genentech and Walter Gilbert? How?

Genentech; they manufactured synthetic DNA

What is Genentech's "insulin factory?"


Which genetically engineered food was the first to be planted on a large scale?


What percentage of the processed food in the United States is genetically engineered?


What was the first state to impose the death sentence as a result of DNA fingerprinting?


Which DNA profiling technique was used in the first death sentence case involving DNA fingerprinting? Why?

Single-locus; less ambiguous and precise allele determination

What three regions of the Romanov skeletal remains were examined?

Teeth, vertebrae, & pelvis

For how many years had the bones in the Romanov burial site been frozen and thawed?

75 years

What type of DNA was used to identify the Romanov skeletons?

Mitochondrial DNA (mtDNA)

Who is the most recent maternal relative for the Romanov family?

Prince Philip, Duke of Edinburgh

Who was Anna Anderson?

Franziska Schanzkowska, a Polish factory worker with a history of mental illness

Cancer of bone, muscle, connective tissue


Cancer of epithelial tissue: Intestinal tract, lung, breast


Mutation in single cell


Inherited in germ line; every cell affected


True/False: Once critical mutation occurs, cancer evolves by accumulating more mutations in genes controlling cell growth


True/False: Cancer is a genetic disease


True/False: Cancer is a fast process


What are the functions of proto-oncogenes?

Promoting cell proliferation & inhibiting cell death (apoptosis)

Mutation in proto-oncogene

Oncogene; cell proliferation "turned on" all the time

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

Signal transduction molecule; oncogene


Transcription factor found in Burkett's Lymphoma; oncogene


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