Molecular Techniques & POCT

assays that target nucleic acid instead of protein, are the latest development in clinical laboratory testing.

requires the release of DNA or RNA from the cell followed by its purification and quantitation.

is a short strand of DNA or RNA of a known sequence that is well characterized and complementary for the base sequence on the test target.

based on the reversible complementary base pairing of nucleotides.

Southern blot Northern blot In situ hybridization Restriction fragment length polymorphism

PCR RT-PCR Transcription based amplification systems Transcription-mediated amplification Nucleic acid sequence-based amplification Strand displacement assay

Probe Techniques-Probe Amplification

Probe Techniques-Signal Amplification

are the simplest types of solid support hybridization assays.

clinical samples are applied directly to a membrane surface. The membrane is heated to denature or separate DNA strands, and then, labeled probes are added.

modification of the dot blot procedure. It was designed to overcome some of the background problems associated with the use of unpurified samples.

In this method, DNA is extracted from a sample using a phenolic reagent and then enzymatically digested using restriction endonucleases to produce DNA fragments.

is a technique that evaluates differences in genomic DNA sequences. This technique can help establish identity or nonidentity in forensic or paternity testing or to identify a gene associated with a disease.

RNA is extracted, digested, electrophoresed, blotted, and finally probed.

for the detection of human papillomavirus (HPV) is a solution-phase hybridization assay that uses an antibody specific for DNA/RNA hybrids to “capture” and detect the hybrids that are formed during the solution hybridization of HPV DNA in the sample with an unlabeled RNA probe.

In this type of setting, both the target nucleic acid and the probe are free to interact in a reaction mixture, resulting in increased sensitivity compared with that of solid support hybridization.

is considered the “gold standard” for many molecular applications from mutation detection to genotyping, but it requires proper methodology and interpretation to prevent misinterpretation.

is a relatively new technique for a short to moderate sequence analysis that is based on the release of pyrophosphate during DNA synthesis as each dNTP (deoxyribonucleotide triphosphate) is incorporated with elongation of DNA.

are very small devices used to examine DNA, RNA, and other substances.

is an amplified hybridization technique that enzymatically synthesizes millions of identical copies of the target DNA to increase the analytic sensitivity.

When the target is microbial RNA or mRNA, the RNA must be enzymatically converted to DNA by reverse transcriptase; the product, cDNA, can then be analyzed by PCR.

allows for direct measurement of amplicon accumulation during the exponential phase of the reaction.

defined as the value at which the sample fluorescence crosses the threshold.

A thermostable enzyme; 5’-3’ polymerase activity