Allele-specific oligonucleotide hybridisation
    Hybridisation of labelled allele-specific oligonucleotides to filter-bound target DNA or the converse.

    Methods for detecting and identifying genomes of microorganisms and mutations or polymorphisms in human DNA. The techniques employ the hybridisation of labelled DNA probes to filter-immobilised target nucleic acid sequences. Identification of variant sequences depends on differences in mobility or hybridisation intensity of DNA fragments hybridising with the probe DNA at a specific temperature and salt concentration.

    Branched DNA (bDNA) assay
    Method based on signal amplification rather than physical amplification of the nucleic acid target. The method uses a very large branched molecule to bind to the target DNA sequence. The branches of the large molecule are then detected using an enzymatic or chemiluminescent method.

    Characterisation of DNA
    To detect changes in DNA, there are four broad categories of tests, which are based on hybridisation, sizing, quantitation and determination of DNA sequence.

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    Denaturing high- performance liquid chromatography (DHPLC)
    A method that relies on a reduced melting temperature of the heteroduplex compared with the homoduplex. DHPLC employs both heat and chemical denaturants to melt DNA. Heteroduplex molecules containing a mismatch will elute from an affinity column before homoduplex molecules, thereby indicating the presence of DNA sequence variation.

    DNA microarrays (colloquially known as ‘DNA chips’)
    Detection of DNA sequences by hybridisation to complementary oligonucleotides or DNA fragments fixed to a solid phase. At the time of publication these are not in routine use but are likely to be integrated into routine diagnostics over the next few years.

    DNA sequencing
    Sequencing uses a variant of the PCR technology with labelled nucleotides and polyacrylamide gel electrophoresis to determine the linear sequence of DNA bases in a PCR product.

    Ligase chain reaction (LCR)
    A method similar to PCR but involving joining DNA primers complementary to the target DNA sequence using a heat-stable DNA ligase enzyme.

    A method to detect single nucleotide variations in amplified DNA, based on the extension of a detection primer that binds immediately adjacent to a variable nucleotide position.

    Multiplex ligation probe amplification
    A quantitative PCR method involving hybridisation and ligation of oligonucleotide probes, followed by PCR amplification of the ligated probes rather than the sample nucleic acids.

    Nested PCR
    Nested PCR improves the sensitivity and specificity of PCR by carrying out two rounds of PCR amplification. The second round of amplification uses primers directed to sequences within the first-round product.

    Nucleic acid amplification techniques
    Include a range of very sensitive methods of detecting, identifying and quantifying minute amounts of nucleic acid (DNA or RNA). Using these techniques it is possible to detect and identify microorganisms, specific genes, mutations and polymorphic DNA, as well as to quantify RNA transcripts or DNA copy number. A number of different nucleic acid detection tests based on nucleic acid amplification are currently in use in Australian laboratories. These include PCR and other nucleic acid amplification techniques.

    Nucleic acid hybridisation tests
    Include a range of methods for the detection and identification of mutations and polymorphic DNA, and for the detection and quantification of microorganisms. A number of different nucleic acid hybridisation tests are currently in use in Australian laboratories.

    Nucleic acid sequence based amplification (NASBA)
    See transcription-mediated amplification (TMA) below.

    Polymerase chain reaction(PCR)
    The first nucleic acid amplification method to be developed. Using primers (short oligonucleotides) complementary to the ends of each strand of the DNA sequence of interest, a heat-stable DNA polymerase is used to extend these primers and create a copy of the DNA. This is cyclically repeated to amplify the sequence millions of times.

    Postamplification techniques
    A group of associated techniques that can identify or characterise amplified DNA or RNA. Techniques include hybridisation, characterisation (sizing and quantification), denaturing high performance liquid chromatography (DHPLC) and DNA sequencing.

    Probe amplification techniques
    These include the branched DNA and Q-beta replicase assays.

    Protein truncation tests
    The protein truncation test is based on the ability to amplify target DNA by PCR and at the same time incorporate into the PCR product a promoter sequence and Kozak consensus sequence. Together, the promoter and Kozak consensus sequence are required for an in vitro transcription and translation assay using the necessary protein machinery present in either rabbit reticulocytes or wheat germ lysates. If a protein truncating mutation is present within the target sequence of interest, the resultant protein will be shorter than expected. Usually, an internal control is present (the wild type sequence) that is used to determine the presence or absence of a premature stop codon by comparison of protein size on a polyacrylamide gel. This assay cannot be used for the identification of missense changes or silent polymorphisms.

    Q-beta replicase assay
    Involves using a modified RNA template, which contains sequences complementary to the target and the enzyme Q-beta replicase. The probe hybridises to the target and can be amplified millions of times.

    Quantitative PCR (Q- PCR)
    An application of PCR to determine the amount of a target sequence in a sample of DNA/RNA.

    Real-time PCR
    A modification in which the PCR product is measured as it is produced, using either binding of a dye or a fluorescent marker. As the rate of rise is proportional to the amount of target, it can be used to quantify the amount of DNA. Examples of this are the commercial LightCycler and TaqMan systems.

    Reverse transcription PCR (RT-PCR).
    As PCR is only able to amplify DNA, detection of RNA involves an initial step of using the enzyme reverse transcriptase to convert RNA into complementary DNA (cDNA) prior to PCR.

    Strand displacement amplification (SDA)
    An isothermal DNA amplification, using target DNA to make cDNA that lacks the restriction enzyme (RE) site. This cDNA is used as a template to bind a primer that contains the RE site, resulting in double-stranded DNA with an RE site at one end of one strand. The RE nicks that strand and the DNA polymerase binds at the nick and then moves along the strand. As it makes a copy of the complementary strand, it displaces the existing homologous strand. This is repeated to create the amplification.

    Transcription mediated amplification (TMA) and nucleic acid sequence based amplification (NASBA)
    Similar isothermal processes that make RNA copies of ribosomal RNA or DNA. A primer binds to target ribosomal RNA and reverse transcriptase is used to make a complementary DNA strand. The cDNA is used as a template to make double-stranded DNA, which is then transcribed by RNA polymerase to multiple copies. Each of these copies can then be used as target RNA to repeat the process.

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