ch. 4 DNA id

What does Cq (or Ct) represent in qPCR?

The cycle at which fluorescence crosses a defined threshold

Which activity of Taq polymerase is responsible for cleavage of the TaqMan probe?

5' exonuclease activity

Why is a melt curve particularly useful with SYBR Green qPCR?

It helps evaluate whether nonspecific amplification products are present

DNA quantitation using agarose gel electrophoresis is particularly useful for:

Small DNA fragments such as PCR products

Why may A320 be subtracted from A260?

To correct for turbidity/background absorbance

Before DNA is immobilized on a slot blot membrane, alkaline treatment is used to:

Convert dsDNA into ssDNA

During TaqMan qPCR, probe cleavage results in:

Separation of the reporter from the quencher

FRET in a TaqMan probe is:

A distance-dependent interaction between reporter and quencher

In colorimetric detection using a biotinylated slot blot probe, streptavidin is linked to:

HRP

What produces fluorescence in a SYBR Green qPCR assay?

Dye bound to dsDNA

In an efficiently amplifying qPCR assay, a 10-fold difference in starting DNA corresponds to approximately:

3.3 cycles

HRP oxidation of TMB produces:

A blue precipitate

Historically, the forensic slot blot assay was used primarily to:

Quantify human genomic DNA

A DNA sample has an A260/A280 ratio of 1.4. This most strongly suggests:

Protein or phenol contamination

A molecular beacon differs from a TaqMan probe because a molecular beacon:

Forms a stem-loop and fluoresces when hybridization opens the structure

Which ratio is particularly useful for detecting residual salts and organic contaminants?

A260/A230

A potential disadvantage of UV transillumination during gel analysis is that UV light can:

Damage DNA

What is a major specificity limitation of SYBR Green compared with TaqMan?

SYBR Green can fluoresce when bound to nonspecific dsDNA products

In qPCR, SYBR Green produces fluorescence primarily when it:

Binds to double-stranded DNA

Compared with UV absorbance, an important advantage of fluorometric DNA quantitation is:

Greater sensitivity at low DNA concentrations

PicoGreen can detect approximately as little as:

20 pg dsDNA

An A260/A280 ratio above 2.0 in a DNA sample may indicate:

RNA contamination

Fluorescent intercalating dyes are useful for DNA quantitation because their fluorescence changes when they:

Bind to DNA

Unlike TaqMan probes, molecular beacons generate fluorescence primarily when the probe:

Hybridizes to its target and the hairpin opens

What is meant by DNA intercalation?

Insertion of a planar molecule between adjacent DNA base pairs

For pure dsDNA, an A260 of 1 corresponds approximately to:

50 µg/mL

Aromatic amino acids and proteins contribute strongly to absorbance near:

280 nm

Which method determines nucleic acid concentration by measuring optical density?

UV absorbance

In an intact TaqMan probe, reporter fluorescence is suppressed primarily because:

The reporter is close to the quencher

Guanidine, chaotropic salts, and some solvents contribute strongly to absorbance near:

230 nm

Which ratio is commonly used to evaluate protein contamination of a DNA preparation?

A260/A280

Which equation corresponds to the UV quantitation method presented in the chapter?

Concentration = (A260 - A320) x conversion factor x dilution factor

Why was the original radioisotope-based slot blot detection method replaced?

Radioisotopes presented safety hazards

What is the purpose of an NTC in qPCR?

Detect amplification arising in a reaction containing no template DNA

A limitation of the D17Z1 slot blot assay is that:

D17Z1 is not completely human-specific because of homology with other primates

Which qPCR detection chemistry produces a signal that is essentially irreversible because the probe is cleaved?

TaqMan

In an ideal qPCR standard curve, a 10-fold decrease in starting DNA shifts the amplification curve approximately:

3.3 cycles to the right

If Sample A crosses the qPCR threshold several cycles earlier than Sample B, Sample A generally contained:

More starting target DNA

Hoechst 33258 is useful for measuring DNA contaminated with RNA because it:

Has little affinity for RNA

Compared with slot blot quantitation, qPCR provides:

Greater sensitivity, objectivity, and automation

Why does TaqMan fluorescence accumulate during successive PCR cycles?

Cleaved reporters remain fluorescent as additional probes are cleaved

A TaqMan probe binds:

Between the forward and reverse primer-binding regions

Which statement correctly compares SYBR Green and TaqMan?

SYBR Green detects dsDNA, whereas TaqMan uses a sequence-specific probe

The D17Z1 probe targets a repetitive alpha-satellite sequence located near the:

Centromere of chromosome 17

In a TaqMan assay, fluorescence increases when:

Taq polymerase cleaves the probe, separating reporter from quencher

In chemiluminescent detection, HRP can oxidize luminol to produce:

Photons

Which qPCR chemistry has the lowest sequence specificity among those compared in the chapter?

SYBR Green

An A260/A230 ratio below 2.0 suggests:

Organic or salt contamination

What happens to quencher suppression when the reporter and quencher become physically separated?

Reporter fluorescence becomes detectable

A DNA sample has an A260/A280 ratio of 1.82. According to the ranges in the chapter, this strongly suggests:

Relatively pure DNA