Chapter 6 Flashcards
Who invented PCR and where did he come up with the idea?
Kary Mullis invented PCR on a highway.
What is the forward primer in PCR?
The forward primer anneals at the beginning of the targeted region of DNA.
What is the reverse primer in PCR?
The reverse primer binds at the end of the targeted region of DNA.
What are the three temperature steps in PCR?
Denaturation-94°C, Annealing-50-60°C, Extension-72°C.
Why is the extension step set at 72°C?
Because of Taq polymerase, which is most active at 72°C.
What is an amplicon?
The PCR product, or amplicon, is the DNA sequence from the 5’ end of one primer to the 5’ end of the other.
:What types of DNA can be used as template DNA in PCR?
Genomic DNA (gDNA), plasmid DNA, or complementary DNA (cDNA).
What is cDNA?
cDNA is a complementary copy of mRNA.
What equipment is required to set up a PCR reaction?
Specialized tubes, plates, and tips.
What are three formats for setting up PCR reactions?
Individual PCR tubes, PCR tube strips, or plates.
What precautions prevent contamination in PCR?
Aerosol-barrier pipet tips, screw-cap tubes, gloves, and designated workstation cabinets with UV lights.
:What is the purpose of negative controls in PCR?
To ensure cross-contamination has not occurred.
Why should PCR reagents be kept on ice before the reaction?
To prevent degradation and unwanted reactions before amplification begins.
What components are combined in a PCR master mix?
DNA polymerase, dNTPs, primers, and reaction buffer.
How is a PCR reaction set up?
A master mix is added directly to template DNA in the PCR tube.
How many target sequences are in genomic DNA versus plasmid DNA?
Genomic DNA has one target in almost 3 billion bp, while plasmid DNA has one target in a few thousand bp.
What type of gel is used to analyze PCR products?
:2-4% agarose gels for products less than 1,000 bp.
What is a thermal cycler?
:A device that rapidly changes temperature to facilitate PCR cycling.
What is ramping speed in a thermal cycler?
The speed at which the block changes from one temperature to another.
What are the sizes of PCR tubes that fit in a thermal cycler?
0.2 ml or 0.5 ml PCR tubes.
How do thermal cyclers change temperature?
By using electricity (Peltier effect) or blowing air over the heating element.
What is a thermal gradient in a PCR machine?
A feature that allows different annealing temperatures to be tested in the same run.
What prevents reaction evaporation in a thermal cycler?
A heated lid or a layer of mineral oil if no heated lid is present
What are the two main types of thermal cyclers?
Conventional PCR instruments and real-time PCR instruments.
What additional feature does a real-time PCR thermal cycler have?
An optical module that detects fluorescence in real time.
What is the advantage of real-time PCR?
It allows quantification of the original template as PCR products are made.
When can PCR products be analyzed in conventional PCR?
Only at the end of the reaction.
How does fluorescence work in real-time PCR?
A fluorescent stain binds to double-stranded DNA, and fluorescence is recorded after each extension step.
What is real-time PCR used for in diagnostics?
Detecting pathogens in human serum or urine, including the Zika virus.
What is digital PCR (dPCR)?
A more sensitive PCR method that allows direct measurement of nucleic acid concentration without standard curves.
What is dPCR used for?
Detecting rare or low-abundance gene targets and small changes in gene expression.
How does digital PCR work?
It divides a sample into many small partitions containing either zero or one (or a few) template molecules, then amplifies each separately.
How are positive and negative samples identified in dPCR?
Positive samples contain amplified product (fluorescent), and negative samples do not (little or no fluorescence).
How is quantitation determined in dPCR?
By the ratio of positive to negative samples.
What enzyme does RT-PCR use?
Reverse transcriptase.
What does reverse transcriptase do in RT-PCR?
It reverse transcribes mRNA into cDNA before PCR begins.
What is cDNA?
The DNA template created from mRNA by reverse transcription.
When is mRNA reverse-transcribed in RT-PCR?
During the first cycle.
What is RT-PCR commonly used with?
Real-time PCR to measure gene expression levels.
How was RT-PCR used in 2009?
It detected RNA viruses in blood, diagnosing influenza A H1N1 cases.
What does multiplex PCR do?
It detects multiple target sequences simultaneously in the same reaction tube.
How does multiplex PCR achieve simultaneous detection?
By using multiple sets of primers that target different sequences.
What is degenerate PCR used for?
Amplifying DNA with limited target sequence information or working with DNA from different species.
What are degenerate primers?
Sets of primers with nearly identical sequences.
How is fast-PCR achieved?
By using specially designed primers and a two-step PCR program combining annealing and extension.
How do isothermal DNA amplification techniques differ from traditional PCR?
They operate at a single temperature instead of cycling through three temperatures.
What is an advantage of isothermal DNA amplification?
It is faster and useful for mobile labs.
What is an example of isothermal amplification?
Loop-mediated isothermal amplification (LAMP).
What does LAMP require?
Multiple sets of carefully designed primers and a strand-displacing polymerase.
What is random amplification of polymorphic DNA (RAPD) used for?
A: DNA fingerprinting when the genomic sequence is unknown.
What does RAPD help identify?
Differences among similar genomes, such as closely related plant species or bacterial strains.
How does RAPD PCR work?
Random primers amplify template DNA, generating PCR products if primers anneal closely in the correct orientation.
How long are RAPD primers?
Ten nucleotides.
How many primers are used in a RAPD PCR reaction?
Four to eight.
What type of polymerase is best for cloning genes using PCR?
A thermally stable DNA polymerase with proofreading ability, such as Pfu.
Where is Pfu polymerase derived from?
The bacterium Pyrococcus furiosus.
When is basic Taq polymerase sufficient?
When PCR is only used to determine the presence of a DNA sequence.
What is InstaGeneTM used for?
Crude genomic DNA extraction.
How does InstaGeneTM prevent DNA degradation?
It contains agents that inhibit cellular DNases.
How is DNA purified for higher purity?
Cells are lysed with detergents, and DNA is purified using a silica spin column.
Why must primer melting temperatures (Tm) be similar?
To ensure efficient and synchronized annealing.
Why should primers avoid intra- or inter-complementary sequences?
To prevent primer-dimer formation.
What is Tm in PCR?
The temperature at which half of the primers dissociate from the template DNA.
Why is Tm important?
It guides the selection of the annealing temperature.
Why should the Tm of two primers be similar?
Because only one annealing temperature can be used.
What is the ideal Tm range?
50–65°C.
What factors affect Tm?
Primer length and base pair composition.
How does GC content affect Tm?
A higher GC proportion increases Tm
What is the ideal GC content for primers?
40–60%.
How do primer-dimers and hairpin loops affect PCR?
They reduce the quantity of primers available for amplification.
Why is magnesium necessary in PCR?
It acts as a cofactor for DNA polymerase.
What are the key cycling parameters in PCR?
Temperature, time at each step, and number of cycles.
What determines the annealing temperature?
The Tm of the primers; it must be determined experimentally.
What affects the temperature of the extension step?
The type of DNA polymerase used.
What affects the duration of each PCR step?
The target sequence length and the processing speed of the polymerase.
What factors influence the number of cycles in PCR?
The quality and quantity of the starting template.
What can DNA microarrays determine?
Which genes in a cell are turned on or off.
What is a DNA microarray?
A solid surface with an array of single-stranded DNA spots representing different gene sequences.
What is the process of making a microarray slide called?
Printing.
How is single-stranded DNA prepared for microarrays?
It is amplified using PCR from a plasmid template and then purified.
How is mRNA processed for microarray analysis?
It is reverse-transcribed into cDNA and tagged with specific colors.
How does cDNA interact with a microarray?
It hybridizes with complementary DNA spots, causing fluorescence.
What has PCR enabled in genome sequencing?
Sequencing entire genomes, including the human genome
How is genome sequencing improving healthcare?
By enabling personalized therapies based on genetic makeup.
How has PCR changed sequencing methods?
It has made sequencing more sensitive and removed the need for radioactivity.
How is cycle sequencing different from PCR?
It uses a single primer and dideoxynucleotide triphosphates (ddNTPs).
What is special about ddNTPs?
Each is linked to a differently colored fluorescent molecule.
Where is cycle sequencing performed?
In a thermal cycler.
How are DNA fragments analyzed after cycle sequencing?
By capillary electrophoresis.
How does capillary electrophoresis detect DNA sequences?
DNA fragments pass a laser, which records them as colored peaks on a chromatogram.
What does each peak in a chromatogram represent?
A single DNA fragment and its fluorescent tag.
What does the order of peaks in a chromatogram indicate?
The DNA sequence from smallest to largest fragment.
What is next-generation sequencing (NGS)?
A method to sequence entire genomes faster, more accurately, and at lower cost.
What is library preparation in NGS?
Breaking genomic DNA into smaller pieces and attaching them to a solid surface.
Why is a DNA fragment library important?
It enables simultaneous analysis of millions of smaller DNA stretches.
What is library amplification in NGS?
PCR amplification of bound genomic DNA fragments.
Why is library amplification necessary?
To ensure the signal generated during sequencing is strong enough for accurate detection.
Amplified DNA fragments are sequenced by what?
one of several different NGS technologies
Sequencing by Synthesis is similar to what?
cycle sequencing
What does Sequencing by Synthesis use to incorporate fluorescent dNTPs one at a time?
DNA polymerase
How are dNTPs in Sequencing by Synthesis labeled?
with a different color
How is the sequence determined in Sequencing by Synthesis?
as the strand is assembled
What is Pyrosequencing based on?
the release of pyrophosphate (PPi) by DNA polymerase during DNA extension
What happens to each dNTP in Pyrosequencing?
it is added one at a time
How is the PPi produced in Pyrosequencing detected?
through chemical reactions that generate light
What is the approach in Pyrosequencing based on?
the release of protons (H+) during the DNA polymerase reaction
What do sequencing microchips do in Pyrosequencing?
position the amplified DNA fragments directly above a semiconductor transistor
What does the semiconductor transistor detect in Pyrosequencing?
changes in the pH of the solution whenever a dNTP is incorporated
What does Sequencing by ligation use instead of DNA polymerase?
DNA ligase
What is added with the ligase in Sequencing by ligation?
various fluorescently labeled sequences
Why can the DNA sequence be determined in Sequencing by ligation?
because ligases are highly specific
What do bioinformaticians apply to data?
the power of computer processing
What types of data do bioinformaticians work with?
DNA sequences, protein sequences, and protein structures
What do bioinformaticians generate?
new information
What is bioinformatics essential for?
to help sort and evaluate data
What is one of the largest databases for DNA and protein sequences?
GenBank
Who operates GenBank?
the National Center for Biotechnology Information (NCBI)
Who funds GenBank?
the U.S. government
What are bioinformatic methods also used for?
to map genomes
What does mapping genomes mean?
to identify important elements such as genes within DNA sequences
What has greatly improved the accuracy and speed of disease diagnosis?
real-time PCR (qPCR)
When is a real-time PCR reaction used?
if a specific disease is suspected
What outbreaks were confirmed by real-time RT-PCR reactions?
the influenza A H1N1 outbreak in 2009 and the Zika virus outbreak in 2015
What is used in genetic modification of plants?
components derived from the common soil bacterium Agrobacterium tumefaciens
What does A. tumefaciens naturally contain?
Ti (tumor inducing) plasmids
What can Ti plasmids do?
insert their DNA into the chromosomal DNA of a plant to cause tumors
What do Bacillus thuringiensis (Bt) bacteria produce?
a delta endotoxin (Cry1)
What is Cry1 lethal to?
specific species of moth larvae
What did scientists use PCR to do with the Cry1 gene?
genetically engineer it into a Ti plasmid
What was the engineered plasmid used for?
to genetically modify crop plants such as corn and soy
What do the GM plants produce?
Cry1
What does producing Cry1 do for GM plants?
makes them resistant to moth larvae such as corn borers
What can be used to identify GM foods?
conventional PCR
What is typically used to quantitate the percentage of GM material in foods?
real-time PCR
What is PCR also used in?
a new type of farming—biopharming
What happens in biopharming?
transgenic animals are created to produce pharmaceutical products such as therapeutic drugs expressed in milk
What is the oldest technique for blood typing?
ABO blood typing
What is a disadvantage of ABO blood typing?
it has a very low power of discrimination
What is an advantage of ABO blood typing?
it is very fast
What has a very high power of discrimination but is time-consuming?
restriction fragment length polymorphism (RFLP) analysis
What is the current method of choice for forensics investigators?
short tandem repeat (STR) analysis
Why is STR analysis preferred?
it is fast and highly discriminatory
What are STR and variable number tandem repeats (VNTR)?
specific regions (loci) of chromosomal DNA that have repeating sequences
What is the size of STR repeating units?
2–6 bp
What is the size of VNTR repeating units?
up to 80 bp
What varies among individuals in STR and VNTR?
the number of repeating units
What is usually different on each chromosome of an individual?
the number of repeats
Why is the number of repeats usually different on each chromosome?
because each member of a chromosome pair is inherited from a different parent
What increases the power of discrimination in STR analysis?
the more loci analyzed
What is the frequency of eight repeats at the TPOX locus in Caucasians?
0.535
That is the chance that a Caucasian has 12 repeats at the TPOX locus?
4.1%
What is the frequency of 12 repeats at the TPOX locus referred to as?
q
What is the probability that a person received the 8 allele from his/her mother and the 12 allele from his/her father represented as?
pq
What have mitochondrial DNA (mtDNA) sequences of modern humans been used for?
to trace the history of mtDNA mutations back to a maternal ancestor—Mitochondrial Eve
Who invented DNA profiling?
Alec Jeffreys
When was DNA profiling invented?
1985
What is DNA profiling based on?
variances in the DNA sequences among individuals
What do genetic variations among individuals sometimes include?
mutations in restriction sites
What happens to restriction sites that have mutations?
they are no longer recognized by the restriction enzyme
When were restriction fragment length polymorphisms (RFLP) discovered?
1978
What does RFLP analysis generate?
a banding pattern unique for each individual
What can the DNA fingerprint generated by RFLP analysis be used for?
to include or exclude a suspect in a criminal investigation
What happens to genomic DNA when performing RFLP analysis?
it is digested with a restriction enzyme
What is used to analyze the fragments generated by restriction enzyme digestion in RFLP analysis?
agarose gel electrophoresis
Why do the fragments appear as a smear rather than as distinct bands on the agarose gel in RFLP analysis?
because there are thousands of restriction enzyme sites within a genome
How are only a select number of DNA fragments visualized in RFLP analysis?
using a technique called Southern blotting
Who invented Southern blotting?
Edwin Southern
When was Southern blotting invented?
1975
What was widely used through the 1980s and 1990s to exclude suspects in criminal investigations?
RFLP analysis and Southern blotting
What is now more commonly used for forensic investigations than RFLP analysis and Southern blotting?
PCR testing
What are disadvantages of Southern blotting?
it requires a large amount of DNA sample and is time-consuming
What are advantages of Southern blotting?
there is no risk of contamination and a positive result is virtually indisputable
What are the basis of the U.S. Federal Bureau of Investigation (FBI)’s Combined DNA Index System (CODIS)?
STR alleles
How many different loci does CODIS use to create a profile of an individual?
20
What must match in parentage testing using STRs?
The number of STR repeats in the child must match the number of repeats on at least one of the father’s chromosomes.
What is the limiting factor in using PCR in conservation efforts?
obtaining funding to support the research
What is unique to every animal or plant species?
their genome
Can human STR sequences be used for other organisms?
no
What have been used to trace human migration and disprove paternity?
Alu elements
