Chapter 7: Amplification by Polymerase Chain Reaction

intrinsic RNase H

The RNA template is then degraded by an ______ activity of reverse transcriptase during the reverse transcription reaction.

Taq polymerase

________ is the most commonly used enzyme for routine PCR applications.

PCR primers

________ are the oligonucleotides that are complementary to the sequences that flank the target region of the template.

parental DNA

The ________ serves as the template for DNA replication.

Universal primers

________ can hybridize to any mRNA sequence in a sample to convert all mRNAs to cDNA.

reagent blanks

Extraction ________, which contain all extraction reagents but no sample, monitor contamination from extraction to PCR.

Reverse transcription

________ is carried out by a reverse transcriptase that forms the basis of reverse transcriptase PCR.

PCR

________ forms the basis of many forensic DNA assays such as DNA quantitation, STR profiling and mtDNA sequencing.

Protein synthesis

also known as translation, is directed by an RNA template.

Melting Temperature

The temperature at which 50% of DNA strands are denatured

Renaturation

The single strands in a solution of denatured DNA can reanneal into double-stranded DNA

anneals

The catalytic function of cDNA synthesis requires a primer that ______ to a complementary mRNA template.

Real-Time PCR

This process allows the monitoring of amplicon production at each cycle of the PCR process

AmpliTaq Gold DNA polymerase

A modified enzyme, remains in an inactive form until activated with a pH below 7 prior to the PCR cycling in which the inhibitory motif is inactivated

Both single

and double-stranded DNA can be used as a template for PCR

Deoxynucleoside triphosphates (dNTPs)

The substrates for DNA synthesis

PCR cycle consists of three elements

denaturation, annealing, and extension

Thermal Cycler

Instrument that carries out PCR amplification

Stochastic Effect

The two alleles in a heterozygous individual are unequally detected at a low level of starting DNA template

Central dogma

The pathway for the flow of genetic information

Reverse Transcription

The flow of genetic information from RNA to DNA

Retroviral RNase H

A domain of the viral reverse transcriptase enzyme

hydrogen bonding

Double-stranded DNA is maintained by _______ between the bases of complementary pairs.

Denaturation

Occurs when the hydrogen bonds of DNA are disrupted, and the strands are separated.

Melting Curve

Can be obtained from measuring DNA denaturation by slowly heat- ing a solution of DNA.

amplicons

The polymerase chain reaction (PCR) allows the exponential amplification of specific sequences of DNA to yield sufficient amplified products, also known as _______, for various downstream applications.

S-shaped amplification curve

An _____ is obtained and divided into an exponential phase, a linear phase, and a plateau.

AmpliTaq Gold DNA polymerase

A modified enzyme, remains in an inactive form until activated with a pH below 7 prior to the PCR cycling in which the inhibitory motif is inactivated.

target sequence

A primer must be specific to the ______ ; otherwise, nonspecific products that might interfere with the proper interpretation of a DNA profile might be produced.

self-complementary sequences

A primer should not contain _______ that may form hairpin structures interfering with the annealing of primers and the template.

primer dimers

These annealed primers may then be amplified during PCR, creating products known as _____, which compete with the target DNA template for PCR components.

Multiplex PCR

is the simultaneous amplification of more than one region of a DNA template in a single reaction to achieve high-throughput analysis.

Divalent cations

are required for the enzymatic activity of DNA polymerases.

Monovalent cations

are usually recommended, and a buffer is often utilized to maintain pH between 8.3 and 8.8 at room temperature.

positive control

A ______ shows that PCR components such as reagents and PCR cycle parameters are working properly during a PCR.

denaturation

At the beginning of each cycle, the two complementary DNA template strands are separated at high temperatures (94°C–95°C) in a process called _______.

annealing

The temperature is then decreased to allow _____ between the oligonuclers and the template. The temperature for annealing is usually 3°C–5°C.

Degradation

causes DNA to break into smaller fragments.

amplified

If the damage occurs at a region to be ______, the result can be a failure in PCR amplification.

Stochastic Effect

The two alleles in a heterozygous individual are unequally detected at a low level of starting DNA template.

internal positive control

The presence of PCR inhibitors can be detected using an _______.

centrifugal filtration

If PCR inhibitors are not eliminated during the extraction process, additional procedures such as the use of _______ devices can be used.

RNA synthesis

With _______ or transcription, the process is carried out using the DNA as a template.

complementary DNA (cDNA)

RNA chains can be used as templates for the synthesis of a DNA strand of complementary sequence, in which the end product is referred to as ______

pol gene

The most common reverse transcriptases used for cDNA synthesis are encoded by the _______ from the avian myeloblastosis virus (AMV) and the Moloney strain of the murine leukemia virus (MMLV).

Oligodeoxynucleotide primers

are an essential component for a reverse transcriptase reaction.

Oligo (dT) primer

can hybridize to the 3′ termini poly (A) tails of eukaryotic mRNAs.

Random hexamer primers

are nonspecific primers that can hybridize, at mul- tiple sites, to any RNA sequence including non-mRNA templates such as ribosomal RNA.

One-step RT-PCR

combines the reverse transcription reaction and PCR in a single tube.

two-step PCR

During a ______, the reverse transcription reaction and PCR are carried out in separate tubes.

end-point PCR method

The ______ measures the amount of amplified product synthesized during PCR at the end of the PCR amplification.

real-time PCR method

With the ______, the amplified product is quantified during the exponential phase of PCR.

hot-start PCR

The ______ strategy is typically used to increase sensitivity, specificity, and yield.