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• amplification of nucleic acids can (obviously ) greatly increase the sensitivity of nucleic acid diagnostics

• developed in 1983 (Kary Mullis) PCR is the most common amplification procedure

• numerous commercial amplification systems are available (i.e. Roche Diagnostics, etc.)

Types of PCR in Diagnostics

Reverse Transcriptase PCR

• converts RNA for amplifiaction:

mRNA --> (reverse transcriptase) --> cDNA --> PCR amplification

Nested PCR

• two stages - amplify whole molecule, then amplify key target

• use 2 sets of primers to get dual (2X) amplification (via two sets of cycles)

• extremely sensitive (due to dual amplification

• weakness - also amplify contaminants (therefore need a very clean sample!

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                 Multiplex PCR                       

• uses two primers with different targets

• can amplify more than one sequence simultaneously

• in diagnostics, can detect multiple pathogens

• can also quantify the amount of DNA / RNA in a specimen

    Broad Range PCR

• uses conserved, specific sequences to diagnose microbial infection (i.e. universal primer)

• can identify pathogens directly  from human blood (without culturing the organisms)

• limited by background contaminants

• commercial system developed by Perkin-Elmer

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    Other Nucleic Acid Amplification Techniques

     T.A.S. (Transcription-based Amplification System)

• uses synthesis of a DNA molecule complementary to the target nucleic acid (usually RNA)

• this is followed by transcription  using cDNA as the template

• amplification of RNA makes it possible to detect RNA-containing viruses

• works using isothermal conditions in a single tube - therefore lowers contamination risks!

• lowers the "detection limit" (increases sensitivity) for certain bacterial / fungal pathogens by using "high copy number" rRNA as targets

• commercial systems are available for M.tuberculosis by Gen-Probe

   

    L.C.R. (Ligase Chain Reaction)

• probes attached to 3' and 5' ends, head-to-tail (end to end)

• get original template, plus a copy from attached

• get a logarithmic increase in products (easily detected by functional groups on the probes

• limits - contamination is also tremendously amplified

• commercial kits available (Abbott Laboratories for STD's)

 

S.D.A. (Strand Displacement Amplification)

• DNA polymerase initiates synthesis at a single-stranded nick and then displaces the single strand

• the displaced single strand acts as a substrate for further nicking and displacement reactions

• procedure is isothermal, uses specific primers, DNA polymerase and a restriction endonuclease (i.e. fairly complex)

• Two impor4tant advantages: (isothermal - except for denaturation, and it works for either single or double stranded DNA)

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    QB Replicase System

• incorporates a single stranded oligonucleotide probe into RNA which is exponentially amplified after target hybridization by the enzyme QB Replicase

• remarkably fast (~ 30 min.) and isothermal

• limit - amplify contaminants - get

• false positives (lately overcome by better target-capture methods)

    Rolling Circle Amplification (RCA)

• requires a circular template and Phi 29 DNA polymerase

• advantage - continuous "circular" amplification

• can produce 10⁷ copies of the original template

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Analysis of Amplification Products

Conventional Method

• amplify target sequence, conduct agarose electrophoresis / EtBr staining, blot / transfer to nitrocellulose / nylon membrane, add labeled probe and view by x-ray film (radioactive probe, or fluorescence)

H.P.A. (Hybridization Protection Assay)

• probe and product incubated together in a single test tube

• probe labeled with an acridinium ester (bound probe protected from alkaline hydrolysis)

• add peroxide and the probe emits light for quantification

• Useful! - don't have to bind DNA to solid matrix, fast (only a few hours), don't have to remove excess unbound probe

D.E.I.A (DNA Enzyme Immunoassay)

• an "anti-DNA" antibody specifically recognizes the hybridization product of "target-and-probe"

• quantify with a colourometric reaction

• accurate and effective!

• commercial products avaialbale (Sorin Biomedica, IncStar, etc.)

Automated DNA Sequencing Technology

• direct sequencing provides rapid, accurate analysis of amplification products!

• Two main approaches: agarose electrophoresis, blot, labeled probes, etc. - or - matrix hybridization (use a solid substrate with antibodies to capture molecule of interest)

S.S.C.P. (Single Stranded Conformational Polymorphism)

• DNA is subjected to PCR with primers for a region suspected of polymorphism (variation ion sequence but with a similar function)

• examine products (which have a marker) after gel electrophoresis

• sensitive enough to detect single nucleotide substitution (has been employed to study resistance of M.tuberculosis)

R.F.L.P. Analysis

• cleave amplified DNA with restriction endonucleases

• gel electrophoresis --> blot to nitrocellulose

• add labeled homologous sequence as probe

• visualize with x-ray film, etc.

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Summary - Nucleic -Acid Based Diagnostics

• many different nucleic-acid based methods

• don't have to culture / grow organism (excellent for dangerous / fastidious organisms)

• can detect disease-causing gene mutations (in humans, etc.)

• can track drug resistance

• fast, sensitive, and improving all the time

• limit - contamination and amplification of contaminants

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