MOL BIO LEC: NUCLEIC ACID AMPLIFICATION

Polymerase chain reaction (PCR)

The first specific amplification method of any type was the?

Target amplification

is a generic term for any method used to increase the copy

number of specific DNA sequence?

Target amplification

involves making many copies of a specific DNA sequence?

Target amplification

This is analogous to growing cells in culture and allowing the cells to replicate their nucleic acid as well as themselves so that, for example, they can be visualized on an agar plate?

Probe amplification

is the number of target nucleic acid sequences in a sample is not changed rather, synthetic probes that are specific to the target sequences bind to the target where the probes themselves are amplified?

Signal amplification

results no change in the number of target or probe sequences; instead, large amounts of signal are bound to the target sequences that are present

in the sample?

Primer

PCR employs DNA polymerase to add a nucleotide to the 3' end of a pre-existing a short DNA fragment called

a _______ to generate new DNA complementary to the template strand?

100 ng - 1 ug

Template or target nucleic acid for PCR?

Primers

are short oligonucleotide sequences that are complementary to the

sequence from the 3' end of the target region of interest for each of the two DNA

template strands?

Deoxynucleotide triphosphate

Building blocks that extend primers to form PCR product?

DNA Polymerase (Taq polymerase)

extends the primers by adding dNTPs

complementary to template to form PCR product?

Reaction buffer

in the form of Mg2+, stabilizes interactions between primers,

template and polymerase which regulates pH optimal enzyme activity?

Mg2+

Reaction buffer is in the form of?

8.5

pH of optimal enzyme activity in PCR?

0.25 mM each primer (oligodeoxynucleotides)

Directs DNA synthesis to the desired region?

0.2 mM each dATP, dCTP, dGTP, dTTP

Building blocks that extend the primers?

50 mM KCl

Monovalent cation (salt), for optimal hybridization of primers to template?

10 mM Tris, pH 8.4

Buffer to maintain optimal pH for the enzyme reaction?

1.5 mM MgCl2

Divalent cation, required by the enzyme?

2.5 units polymerase

The polymerase enzyme

that extends the primers (adds dNTPs)?

10 2-10 5 copies of template

Sample DNA that is being tested?

Denaturation

Annealing

Extension

What are the elements of a PCR cycle?

Denaturation

Separation of a double stranded DNA template into two single strands?

Single stranded

For DNA synthesis to occur, the DNA template must be?

95 °C

Denaturation

The reaction temperature is increased to _____ which melts (disrupts the hydrogen bonds between complementary bases) all dsDNA into single-stranded DNA (ssDNA)?

Annealing

Hybridization of DNA primers to the DNA template strands?

Annealing

The primers bind

to the target DNA sequences within the template strands in a sequence-specific

manner, following the rules of base-pair complementarity?

5 °C below

Annealing

The temperature is lowered to approximately ________ the melting temperature (Tm) of the primers (often 45-60 °C) to promote primer binding to

the template.

Extension

Addition of nucleotides to the primers by DNA polymerase?

Extension

Once primers

bind to the templates, a DNA polymerase reads the template sequences and adds

complementary nucleotides to the 3' ends of the primers, generating longer DNA

strands?

72 °C

Extension

The temperature is increased to _______, which is optimum for DNA polymerase activity to allow the hybridized primers to be extended?

1000 bases in 1 minute

Under optimum conditions,

Taq polymerase extends?

Positive control

What control

Known sample containing target sequence?

Positive control

Ensures that DNA polymerase enzyme is active

Positive control

What control

Primers are annealing to correct sequence?

Positive control

What control

Thermocycler is working properly

Positive control

What control

Buffer is optimal

Blank control or reagent control

Reaction without DNA added to ensure that reagent mix is not

contaminated with template or previously amplified PCR product?

Negative template control

DNA sample known to lack target to ensure that primers do not anneal to

unintended sequence?

Internal control or amplification control

is a second primer set for a

sequence unrelated to target sequence of interest but present in all samples tested.

Internal control or amplification control

What control

It can be performed in same tube or can be run as a duplicate sample.

Internal control or amplification control

What control

Ensures that DNA sample does not contain inhibitors and reaction mix is working properly

Internal control or amplification control

What control

Distinguishes between true negative (sample without target sequence) and false negative (amplification failure) results

Internal control or amplification control

are used with qPCR assays to detect false-negative results in

the event of amplification failure?

RNA

In Internal control or amplification control, these controls should be ____ for RNA expression assays?

Plasmid

In Internal control or amplification control, these controls should be ____ for DNA copy-numbrr analyses?

uracil

The system uses PCR mixtures with dUTP rather than TTP; therefore, amplicons

contain _____ rather than thymidine residues?

50°C for 2-10 minutes

Chemical: dUTP-UNG system (AmpErase® system)

The mixture is incubated at __________ to eliminate any contaminating amplicons from previous reactions?

Native DNA

_______ lacks uracil and is immune from degradation?

Hot start polymerase chain reaction (PCR)

The purpose of __________ is to optimize the

yield of the desired amplified product in PCRs and, simultaneously, to suppress

nonspecific amplification and formation of primer dimers

Hot start polymerase chain reaction (PCR)

This is achieved by

withholding an essential component of the PCR-the DNA polymerase, or the primers, for example-until the reaction mixture has been heated to a temperature that inhibits

hybridization of primers to one another or to nonspecific regions of the template.

Touchdown PCR

Is a method to decrease off-target priming and hence to increase the specificity of PCRs.

5°C-10°C higher

In touchdown PCR the temperature selected for the annealing

step is initially set ______ than the calculated Tm of the primers

Touchdown PCR

essential when the sequence of the primer

might not match that of the target-for example, if the sequence of the primer has

been deduced from amino acid sequences, when the template DNA may contain

several closely related targets, or when the target DNA is of a different species from

that used to design the primers?

Conventional PCR

the amplified DNA product, or amplicon, is detected in an end- point analysis?

Real-time PCR

the accumulation of amplification product is

measured as the reaction progresses, in real time, with product quantification after each cycle?

Real-time PCR

is enabled by the inclusion of a

fluorescent reporter molecule in each reaction well that yields increased

fluorescence with an increasing amount of product DNA?

DNA-binding dyes and fluorescently

labeled sequence-specific primers or probes

The fluorescence

chemistries employed for Real-time PCR include?

Specialized thermal cyclers

___________ equipped

with fluorescence detection modules are used to monitor the fluorescence signal as

amplification occurs?

Threshold

the point at which significant and specific amplification occurs and the fluorescence signal rises above the background level?

Threshold cycle (ct/cq)

the cycle number at which the fluorescence signal

crosses the threshold?

Linear ground phase

What phase

where PCR begins. The amplification is not detectable?

Early exponential phase

What phase

where the exponential amplification becomes

detectable?

Early exponential phase

What phase

The DNA amount doubles at each cycle?

Early exponential phase

What phase

Quantitation of nucleic acid must be done at this phase.

Log-linear phase

What phase

where reaction slows down due to the depletion of reagent

components and lowered PCR efficiencies

Plateau phase

What phase?

where the reaction components become limited and the

reaction stops?

Plethora

A ______ of fluorescence-based technologies have been developed to support the popularity of

the assay.

Fluorescence dye-based detection

This method utilizes a dye that emits

fluorescence when incorporating itself into double-stranded DNA?

Fluorescence dye-based detection

The fluorescence signal increases at each PCR cycle as more double-stranded DNA

molecules are generated?

SYBR® Green

The most common dye used in fluorescence dye-based detacrion is?

Fluorescent probe-based detection

Fluorescently-labeled oligonucleotide probes detect specific DNA sequences

TaqMab probes

recognize a specific region within the target DNA sequence?

fluorophore

Fluorescent probe-based detection

The probes are labeled with a _______ at the 5' end and a quencher at the 3' end.

quencher

Fluorescent probe-based detection

The

probes are labeled with a fluorophore at the 5' end and a ______ at the 3' end.

Fluorescent probe-based detection

The 5'-3'

exonuclease activity of the polymerase cleaves the probe, releasing the

fluorophore away from the quencher, thus resulting in increased fluorescence

FRET probes

bind to the PCR product in a sequence-specific fashion?

two fluorescence resonance energy transfer (FRET)

When _______ probes bind to the targets in close

proximity, the energy transfer from the donor fluorophore to the acceptor

fluorophore takes place allowing emission of fluorescence from the acceptor

FRET probes

This fluorescence signal is observed during the annealing step?

PCR

relies on the ability of DNA polymerase to synthesize a new DNA strand from

a DNA template.

complementary DNA (cDNA)

When the nucleic acid of interest is RNA, an extra step is required initially

to generate a _________________- template from the RNA.

reverse transcription

When the nucleic acid of interest is RNA, an extra step is required initially

to generate a complementary DNA (cDNA) template from the RNA. This step is called

________________

reverse transcription PCR

The PCR analysis of RNA is therefore referred to as

reverse transcription PCR

is very sensitive and commonly used to detect and quantify messenger

RNA (mRNA).

One-step assay

RTPCR

One-step assay / two-step assay?

combine reverse transcription and PCR in a single tube and buffer, using

a reverse transcriptase along with a DNA polymerase.

One-step assay

RTPCR

One-step assay / two-step assay?

only utilizes

sequence-specific primers.

Two-step assays

RTPCR

One-step assay / two-step assay?

is recommended in the analysis of large numbers

of samples,

Two-step assays

RTPCR

One-step assay / two-step assay?

involves multiple analysis of targets in one sample.

Two-step assays

RTPCR

One-step assay / two-step assay?

reverse transcription and PCR steps are performed in separate tubes,

Two-step assays

RTPCR

One-step assay / two-step assay?

different optimized buffers, reaction conditions,

and priming strategies.

Quantitative reverse transcription PCR (RT-qPCR)

is used in a variety of

applications including gene expression analysis, RNAi validation, microarray

validation, pathogen detection, genetic testing, and disease research.

Quantitative reverse transcription PCR (RT-qPCR)

When designing this assay it is important to decide whether to use

total RNA or purified mRNA as the template for reverse transcription.

total RNA

is often used in

Quantitative reverse transcription PCR (RT-qPCR) because it has

important advantages over mRNA as a starting material.