Chapter 9: Detection Methods

Fluorescent Intercalating Dye Staining

Fluorescent Intercalating Dye Staining

These techniques allow the detection of DNA bands as small as 10 ng in agarose gels.

ethidium bromide-stained

Historically, ________ gels were photographed with a standard ultraviolet transilluminator at approximately 300 nm using a camera with an orange filter, whereas gels can currently be documented using digital techniques.

Ethidium bromide

Is a mutagen and a potential carcinogen. It should be handled according to the Material Safety Data Sheet and safety protection wear should be used while handling the chemical.

Ethidium bromide waste

is usually disposed of as hazardous waste and is handled in accordance with laboratory guidelines.

Silver staining

________ of polyacrylamide gels has been used for the amplified fragment length polymorphism method of variable number tandem repeat profiling.

100 times

The sensitivity of silver staining is approximately ______ higher than that obtained with ethidium bromide, and silver staining is less hazardous than ethidium bromide detection.

Radioisotope probe labeling

was used for early versions of VNTR testing and DNA quantitation.

Nick translation

incorporates labeled deoxyribonucleotides (dNTPs) into double-stranded DNA.

DNA Polymerase I

Recognizes the nicks and replaces the preexisting nucleotides with new strands containing labeled dNTPs.

single-stranded fragments

Prior to hybridization, the probe is denatured into ______ by boiling for a few minutes followed by rapid cooling on ice.

alkaline phosphatase (AP)-conjugated probes

The use of _______ with chemiluminescent substrates comprises a highly sensitive non-radioisotopic detection system.

Alkaline phosphatase

can cleave the phosphate groups from a variety of substrate molecules.

Lumi-Phos Plus kit of Lumigen Inc.

The _______ contains this substrate and can serve as a detection system for slot blot assays for DNA quantitation and RFLP assays for VNTR profiling.

Biotin

Also known as Vitamin H, a water-soluble molecule found in egg yolk.

Avidin

A glycoprotein found in egg white; it binds to biotin with extremely high affinity.

nonspecific binding

Avidin detection has a high background due to _______.

Reporter Enzyme Assay

This technique has been used for forensic DNA testing such as slot blot assays for DNA quantitation.

Fluorescent Dyes

They have the capacity for simultaneous analysis of complex samples such as multiplex PCR products with different fluorescent labels allowing the distinction of various amplicons.

Labeling Methods

Fluorescent dye labeling can be incorporated into a DNA fragment using a 5′-end fluorescently labeled oligonucleotide primer.

Fluorophore

A component of a fluorescent dye molecule that causes the molecule to be fluorescent.

argon ion gas laser

The ______ is frequently used in applications such as fluorescence-labeled STR and mtDNA sequence analysis because the excitation wavelength of commonly used fluorescent dyes matches the wavelength of the argon laser.

Optical filters

are used to filter out undesired light and to allow only one particular wavelength to pass through.

excitation filter

The _______ selectively transmits light from an excitation source.

dichroic beam splitter

The light is then directed by the _______ to DNA molecules labeled with fluorescent dyes.

emission filter

The ______ selectively blocks undesired light, thus transmitting a specific wavelength of the emitted fluorescence.

arbitrary unit

The signal from the fluorophore is collected and converted to an electronic signal expressed in an ______ such as a relative fluorescence unit (RFU).