Chapter 6: DNA Quantitation

alkaline solution

alkaline solution

Prior to the quantitation, an _______ is added to the genomic DNA sample, which denatures DNA.

D17Z1 probe

Labeled with radioisotopes that could be visualized by exposing the slot blot membrane to x-ray film.

nitrocellulose membrane

Generating single-stranded DNA is necessary for DNA to be cross-linked onto a _____.

Alkaline phosphatase-labeled probe

It can be coupled with chemiluminescent detection.

Biotinylated probe

It can be coupled with either colorimetric or chemiluminescent detection.

colorimetric detection

In ______, the biotin moiety of the probe is bound to streptavidin.

horseradish peroxidase

The streptavidin is conjugated with ______, which catalyzes the oxidation reaction of tetramethylbenzidine (TMB)

chemiluminescent detection

The sensitivity of _____ is slightly higher than that of colorimetric detection.

Quantitative measurements

_____ can be made by comparing an unknown sample to a set of standards with known DNA concentrations.

Intercalating dyes

______, usually planar molecules, can slide themselves in between base pairs of DNA without breaking the DNA double helix.

Quant-iT PicoGreen dsDNA reagent (Invitrogen)

It is a fluorescent intercalating dye that stains double-stranded DNA (dsDNA) for quantitation in a sample. The detection limit of this method is approximately 250 pg.

standard spectrofluorometer

DNA samples are simply added to a solution containing the fluorescent intercalating dye. The fluorescence, proportional to the quantities of DNA is measured using a ______ with excitation and emission wavelengths of the light source.

standard curve

A ______ is first created using samples containing known amounts of DNA.

End-point PCR methods

______ measure the quantity of amplified product at the end of PCR.

Real-time PCR methods

_______ can quantify the amplified DNA during the exponential phase of PCR.

Real-time quantitative PCR (qPCR)

It was developed in the early 1990s, and it analyzes the amplification of a target sequence at each cycle of PCR.

Fluorescent reporter

Used to monitor the accumulation of amplified products during PCR.

TaqMan Method

This method utilizes the 5′ exonuclease activity of Taq polymerase to cleave the probe during PCR.

minor groove binder (MGB)

A ______ is often linked at the 3′ ends of the probe.

conjugated MGB

A ______ binds to the minor groove of a B-form DNA helix which is stabilized by Vander Waals forces.

oligonucleotide probe

The ______ is labeled with both a reporter fluorescent dye, and a nonfluorescent quencher moiety.

Fluorescent Resonance Energy Transfers (FRET)

A distance-dependent interaction between two molecules in which the excitation energy is transferred from a photon donor molecule (reporter) to an acceptor molecule (quencher) without emission of a photon.