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