Molecular Testing and Clinical Diagnosis Amplified Nucleic Acid Testing

Nucleic Acid Amplification: Culture Analogy Reproduce a single, organism colony forming unit to produce enough organism to perform identification testing Nucleic Acid Amplification Nucleic Acid Staphylococcus sp. single organism Subculture to reproduce more organism for testing An analogy can be drawn between nucleic-acid amplification and microbiological culture techniques. The purpose of culture is to grow enough of the organism so that a positive identification can be made. In the case of culture identification probe assays, the organism is grown to reproduce or amplify enough nucleic acid so that the DNA probe can identify the organism with high sensitivity. In the case of nucleic acid amplification, essentially the same thing is occurring in an artificial manner. These techniques enzymatically amplify, or copy, the nucleic acid in a test tube to a very high level so that DNA probes can be used to easily identify the resulting copies of nucleic acid or amplicon. Detect specific amplified or copied nucleic acid

Nucleic Acid Amplification Key Principle: nucleic acid amplification techniques enzymatically amplify (multiply) a specific nucleic acid sequence, exponentially producing billions of copies in a short period of time Amplification needed if the patient’s sample contains too few copies of the nucleic acid being testing for Although nucleic acid probe assays are very sensitive and specific, addition of a nucleic-acid amplification step can increase the sensitivity. Nucleic-acid amplification techniques enzymatically amplify, or multiply, a specific nucleic acid sequence exponentially, resulting in the production of billions of copies in a short period of time.

Advantages of Nucleic Acid Amplification Assays High sensitivity Fast turn around times Examples of uses: Detect unculturable/slow growing organisms Detect small quantities of nucleic acid Detect mutations in genetic sequences/tumor markers/inherited diseases Human identity (forensics) It has been well documented that amplification assays are usually more sensitive than culture and enzyme immunoassays. In addition, microbial-identification results can often be obtained much faster by amplification methods than by the corresponding culture method. For example, the Gen-Probe MTD assay can definitively detect Mycobacterium tuberculosis in respiratory specimens in four hours or less – compared with up to six weeks for culture. Amplification assays can also detect organisms that are difficult or impossible to grow in culture.

Amplified probe assays have four main steps Sample preparation Amplification Hybridization Detection

Amplified Assays Utilize Enzymes to Amplify Target DNA or RNA DNA polymerase - copies DNA into DNA Reverse transcriptase - rewrites RNA into DNA RNA polymerase - binds double stranded DNA and transcribes into RNA DNA ligase - joins together double stranded DNA fragments

Polymerase Chain Reaction Gold standard in amplification techniques Can be used with DNA or RNA starting material RNA must be reverse transcribed first (RNA rewritten into DNA)

Three Cycle Steps 1. Denature (94ºC) double stranded DNA into single strands with heat 2. Anneal (55ºC) oligonucleotide primers to target DNA 3. Extension (72ºC) DNA polymerase extension of primers to create complementary DNA strand Cycles: steps are repeated (n) times generating 2n number of amplicons Amplicons are then detected

Components of PCR Reaction Template nucleic acid must be DNA (the unknown in the patient sample) Thermo-stable DNA polymerase Two oligonucleotide primers (5` and 3`) dNTP’s Reaction buffer Magnesium chloride

Oligonucleotide Primers Short pieces of synthetic DNA 10-30 nucleotides in length Select sequences with 50-60% Guanine-Cytosine content to obtain good binding Provide assay specificity

dNTP’s - Deoxynucleotides Nucleotides are needed to synthesize strands of nucleic acid (building blocks)

Reaction Buffer Salts Buffered pH Promote enzyme activity- thermo-stable DNA polymerase

Magnesium Chloride Required for thermo-stable DNA polymerase activity Cofactor for the enzyme

Detection of Amplicons Electrophoresis (conventional or capillary) Agarose gel Polyachrylamide Hybridization to probes that have detection device (radioactive isotopes, fluorophore, enzyme) Incorporation of fluorescent tags during amplification Electrophoresis – both DNA and RNA are negatively charged and will migrate toward the anode (the positively charged electrode). Separation of different nucleic acids occurs when mixtures are allowed to travel through a neutral sieving polymer under the electrical field. Separation is primarily based on molecular weight, with smaller molecules traveling faster through the polymer than larger ones. Agarose polymers are cast in trays and submerged in buffer. The gels are permeable to fluorescent nucleic acid-binding dyes and results of electrophoresis are recorded by a photographic image of a stained gel. Polyacrylamide polymers are suited for high-resolution separation. Polyacrylamide is used either as a linear polymer solution, which is filled in capillaries (capillary electrophoresis) or as cross-linked gels, which are cast between two plastic or glass plates. These gels are also permeable to fluorescent stains. Capillary electrophoresis (CE) is carried out in a small-bore fused silica capillary tube. In contrast to conventional electrophoresis, CE is well suited to automation. Samples are easily applied to the capillary, a variety of detector types can be used, and the resulting electrophoretograms can be analyzed and manipulated in much the same manner as chromatograms. Probe detection devices are similar to those used in immunologic methods (enzyme immunoassays, fluorescent-labeled immunoassays). Radioactive probes have a short half-life limited by isotopic decay. This inherent instability, along with concerns of radioisotope safety and disposal restrict the use radioactive proves in clinical laboratories.

Real-time PCR Real-time detection – data is collected during nucleic acid amplification rather than at a particular endpoint. Fluorescent reporter molecules detected during thermal cycling Quantitative (how much was in the patient sample) All steps performed in same reaction tube – no sample transfers decreases risk of contamination in subsequent reactions Quick turn-around time: replacing many conventional techniques in the clinical laboratory

Other Amplification Techniques Variations of the PCR process Examples: Ligase chain reaction – 2 probes attach to single stranded DNA, DNA ligase joins probes together (instead of extension with nucleotides) to form DNA hybrid Transcription mediated amplification – RNA transcription amplification Many commercial systems have made variations in the ‘classic’ PCR amplification procedure. There are many variations. Examples of two variations. Ligase chain reaction (LCR) is a probe-based amplification system. The procedure is similar to PCR in that temperature is used to melt DNA apart. With LCR, much longer primers are allowed to anneal to the target DNA. Two primers or probes attach to a single DNA strand. The two primers attach in close proximity to each other. A DNA ligase enzyme joins the two primers to form the complementary DNA strand. In PCR, the complementary DNA strand is formed by extension of one primer with the addition of individual nucleotides. The LCR primers have the label or detection system. Transcription mediated amplification (TMA) is a RNA transcription amplification system using two enzymes to drive the reaction: RNA polymerase and reverse transcriptase. The entire reaction is performed at the same temperature (unlike PCR). TMA can amplify either DNA or RNA and produces RNA product, or amplicon, in contrast to most other nucleic acid amplification methods that only produce DNA. TMA has very rapid kinetics resulting in ten-billion-fold amplification within 15 to 30 minutes. TMA has been combined with HPA detection in a single-tube format, making the procedure relatively simple to perform.

Summary: Amplification Methods Much like a culture technique, they increase likelihood of detection and identification Enzymes are used to increase target sequence for detection May be automated or semi-automated more easily if isothermal

Summary Amplification Methods Increased sensitivity amplification Specificity primers probe/detection systems