Molecular Testing and Clinical Diagnosis
Amplified Nucleic Acid Testing
CultureReproduce a single, organism colony forming unit to produce enough organism to perform identification testing
Nucleic Acid Amplification: Culture AnalogyNucleic Acid Amplification
Staphylococcus sp. single organism
Subculture to reproduce more organism for testing
Nucleic Acid
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
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)
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 Steps1. Denature (94ºC) double stranded DNA into
single strands with heat2. Anneal (55ºC) oligonucleotide primers to
target DNA3. 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
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
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