| Terms and Definitions Term Definition Codon A three nucleotide base sequence that codes for an amino acid Genome The genetic code composed of 64 codons that code for 21 amino acids and 3 stop codons. (amino acids are the building blocks of proteins and stop codons stop the writing process much like a period at the end of a sentence) Nucleic acid Polymer made of monomers; two examples are RNA and DNA Transcription Process of transferring information from DNA into an RNA message Translation The formation of an amino acid from RNA Deoxyribonucleic Acid (DNA) A double-stranded polymer of nucleotides that houses genetic information Ribonucleic acid (RNA) Typically a single-stranded polymer that is much shorter than DNA but chemically similar with a few differences (e.g.- uracil replaces thymine). Replication Reproduction of DNA content from parent to daughter cell during cell division Amplification methods Techniques that increase the amount of the target, the detection signal, or the probe so that sequences are readily detected Fluorescence The emission of light at a longer wavelength when the light is excited at a shorter wavelength Oligonucleotide Short single-stranded nucleic acid Probe A nucleic acid used to identify a hybridization target Polymerase Chain Reaction (PCR) An amplification method performed in vitro | View Page |
| Classification Molecular methodologies are as varied as the targets they are created to seek, but for the purposes of this module, they will be broadly separated into those that require no amplification, or direct tests, and those that employ amplification or copying, thus termed amplified tests. | View Page |
| Overview As was earlier stated, molecular methodologies are as varied as the targets they are created to seek and there are many ways to arrive at the same answer. There are many combinations of target/amplification/detection.This module will briefly explain the specific uniqueness of each approach. Advantages and disadvantages of each will not be discussed as these will vary by lab, patient population, and other factors discussed in the clinical application section. | View Page |
| Which of the following is not an example of an amplification method? | View Page |
| Advantages of Molecular Testing Molecular methodologies offer numerous advantages to the clinical laboratory. These include:Sensitivity: Amplification methodologies are particularly useful in increasing the sensitivity of a methodology and useful in the identification of target molecules of interest that are only present in low concentrations. Specificity: Molecular methods minimize false positive test results by targeting the specific molecule of interest.Turn Around Time: In comparison with standard traditional culture methods, molecular methodologies usually offer better turn around times from receipt to result reporting.Application: broader application can be found with molecular methodologies such as infectious diseases, genetic testing, forensics, drug resistance, and tumor marker detection and monitoring. | View Page |
| Disadvantages of Molecular Testing Molecular methodologies while highly advantageous do contain limitations and certain disadvantages. These can include:Cost: Molecular methodologies are usually more expensive than standard traditional methodologies. Equipment and reagent costs could be prohibitive to some laboratories. As molecular methods become more standard, the costs could potentially decrease. Currently, laboratories that consider the cost prohibitive prefer to transport molecular specimens to a reference laboratory.Personnel requirements: Depending on laboratory accreditation requirements and testing methodologies some personnel may not be qualified to competently perform molecular testing. Laboratory space requirements: Molecular amplification methods require dedicated space that may not be available in some clinical laboratories. | View Page |
| Which of the following are considered advantages of molecular testing? | View Page |
| Direct Nucleic Acid Testing Principle These methodologies use principles that detect RNA or DNA that is currently available in the sample; therefore no multiplication or amplification occurs. There are usually 3 main steps: Sample preparation Probe hybridization Detection | View Page |
| Amplified Nucleic Acid Testing Principle These methodologies use principles that amplify or multiply the target of interest, usually incorporating an enzyme to produce millions or billions of copies in a relatively short time.Some enzymes used in amplification include: DNA ligase DNA polymerase RNA polymerase Reverse transcriptase Alkaline phosphatase Cleavase Note: the steps in amplified testing will vary depending on the target amplified, requirement for thermal cycling and detection techniques. | View Page |
| Amplification As seen in the preceding table of amplified nucleic acid test methodologies, any of the following can be amplified: Target (most common) Probe Signal When employing an amplification procedure, each methodology can differ based on: Amount of target Amplification type Enzyme requirements Thermal cycling (thermocycling) requirements Detection methodology | View Page |
| Amplification Issues The product of an amplification reaction is termed an amplicon. Amplicons are the outcome of what is to be detected. Due to the sensitivity of amplification methodologies, and the nature of the amplicon, just a small amount of contamination can cause false positive results.Quality control of these various steps in the analytical procedure assure minimal contamination and quality results. | View Page |
| Amplified Nucleic Acid Tests Amplification Method Amplifies Use of Thermal Cycling (Thermocycling) Polymerase Chain Reaction (PCR) Target amplification using DNA polymerase Yes Ligase Chain Reaction (LCR) Target amplification using DNA ligase Yes Transcription- based or Transcription-mediated amplification(TMA) Target amplification using reverse transcriptase and RNA polymerase No Strand Displacement (SDA) Target amplification using DNA polymerase that continuously displaces strands of DNA containing the target sequence No Branched DNA (bDNA) Signal amplification using alkaline phosphatase No Loop Mediated (LAMP) Target amplification of multiple DNA sequences in a loop pattern using DNA polymerase No Nucleic acid sequence based (NASBA) Target amplification using 3 enzymes No Q-beta Replicase Probe amplification- The concentration of an RNA probe increases if the target is present No | View Page |
| Which of the following steps is not included in a direct nucleic acid test? | View Page |
| Match the following tests to their appropriate principle: | View Page |
| Thermal Cycling Thermal cycling (thermocycling) uses specific instrumentation to cause variations in temperature during an amplification procedure in order to produce copies of the target of interest (or amplify the target of interest) so that detection can take place.The temperature variations increase to unfold the DNA and then decrease to allow for reconnection or reannealing. | View Page |