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Molecular Diagnostics Final Exam

Includes material from exams 3 & 4
STUDY
PLAY
What are the 5 components of PCR?
-Primers
-Taq Polymerase
-Buffer
-DNA Target
-Deoxynucleotide triphosphates (A, C, T, G)
Short synthetic oligonucleotides consisting of 20 to 30 bases in length that start the PCR reaction
Primer
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?
Magnesium
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.
Doubles
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?
1
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
Amplicon
To decrease the possibility of DNA cross-contamination, workflow in a molecular lab is ______________?
Uni-directional
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?
No
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
One
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 _____________?
Semi-quantitative
Real-time PCR is ___________?
Quantitative
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
Exponential
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?
-Taqman
-Molecular Beacon
-Cleavase Invader
Which fluorescence method uses a hydrolysis probe?
TaqMan
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?
-TaqMan
-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
Billions
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?
Cleavase
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?
Proportional
What percentage of DNA is different between humans to distinguish individuals?
0.1%
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?
Non-coding
Are STRs in the coding or non-coding regions?
Non-coding
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?
PCR
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
Multi-locus
Which portion of the genome (intron/exon) are the VNTRs/STRs located?
Intron
Combined DNA Index System; a federally maintained database of DNA obtained from crime scenes and convicted violent offenders
CODIS
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?
2pq
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?
Circular
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?
Males
Y chromosomes are inherited from which parent?
Father
Power of Discrimination and speed of analysis:
RFLP Analysis
-High discrimination
-Slow speed
Power of Discrimination and speed of analysis:
mtDNA
-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?
Yes
Is DNA transferred from one species to another functional?
Yes
Did scientists at the Asilomar Conference in California set guidelines for genetic engineering?
Yes
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?
No
In the United States, can living things be patented?
Yes
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?
Insulin
Who won the race between Genentech and Walter Gilbert? How?
Genentech; they manufactured synthetic DNA
What is Genentech's "insulin factory?"
Bacteria
Which genetically engineered food was the first to be planted on a large scale?
Tomatoes
What percentage of the processed food in the United States is genetically engineered?
70%
What was the first state to impose the death sentence as a result of DNA fingerprinting?
Florida
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
Sarcoma
Cancer of epithelial tissue: Intestinal tract, lung, breast
Carcinoma
Mutation in single cell
Sporadic
Inherited in germ line; every cell affected
Familial
True/False: Once critical mutation occurs, cancer evolves by accumulating more mutations in genes controlling cell growth
True
True/False: Cancer is a genetic disease
True
True/False: Cancer is a fast process
False
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
ras
Transcription factor found in Burkett's Lymphoma; oncogene
myc
Inhibits signal transduction; tumor suppressor gene; leads to colorectal carcinoma
APC
Involved in DNA repair; tumor suppressor gene; leads to familial breast & ovarian carcinoma
BRCA
Alters activity of transcription factors & controls cell division; tumor suppressor gene
Rb (Retinal blastoma)
Genetic abnormality; translocation of Chromosomes 9 & 22
Philadelphia Chromosome
Condition associated with Philadelphia Chromosome
Chronic Myelogenous Leukemia (CML)
What gene is responsible for the translocation of chromosomes in CML?
Abnormal BCR-ABL gene
How does Gleevec control CML?
Inhibits BCR-ABL gene & induces apoptosis in BCR-ABL positive cells
What lab test is used to identify an abnormal BCR-ABL gene?
FISH
What is the function of the BRCA gene?
DNA repair & helps maintain integrity of genomic DNA
What percentage of breast cancer cases are familial?
10%
What percentage of familial breast cancer cases are attributed to a mutation in the BRCA gene?
75%
Genetic inheritance of the BRCA mutation is _______ ______?
Autosomal dominant
How does a mutation in the BRCA gene cause breast cancer?
Tumor suppressor gene is lost; over time leads to malignancy
What is the incidence of ovarian cancer associated with a mutation in the BRCA gene?
90%
Is James Watson a controversial character?
Yes
Is manic depression considered a genetic disease?
Yes
Have mice been genetically engineered?
Yes
How have mice been genetically engineered?
Human genes have been synthesized and inserted into mice
What percentage of genetic material do humans and mice have in common?
99%
How many genetic diseases are known?
5,000
What is the relative number of individuals carrying a cystic fibrosis gene?
1 in 25
Did Mary-Claire King and Mark Skolnick work closely together to find the "breast cancer gene?"
No; they were competing
What source did Mark Skolnick use to study "breast cancer" families?
Mormon family history library
How did Mary-Claire King find "breast cancer" families for her study?
Television news announcement
On which chromosome was the first "breast cancer" gene found?
Chromosome 17
The drug Gleevec was developed to treat which disease?
Chronic Myelogenous Leukemia (CML)
How many women in the U.S. are thought to carry the "breast cancer" gene?
600,000
Which technique compares normal and cancer cells?
Micro-array