Molecular methods Flashcards
Study of proteins on the cellular level which could provide information about disease process, environmental conditions, drug effectivity, etc.
Proteomics
Study of DNA of an organism to characterize it further in molecular level.
Genomics
Study of microbial genes from the environment and human normal flora that may affect human health, metabolism, nutrition, and immune function. The study is intended for non-culturable microorganisms, since only 1% of all prokaryotes on Earth are culturable in the laboratory.
Metagenomics
A DNA sequence that encodes a specific product.
Gene
Comprises all genes in an organism.
Genome
A single, unpaired (haploid) circular dsDNA molecule that contains bacterial genetic information.
Bacterial Chromosome
dsDNA, closed, circular autonomous extrachromosomal genetic element.
Plasmid
Extrachromosomal mobile genetic elements.
Mobilome
A form of cell division; mode of bacterial replication.
Binary fission
DNA polymerase enzyme; STEPS: Unwinding/Relaxation of DNA → Separation → Synthesis → Termination.
DNA Synthesis
UAA, UAG, UGA.
STOP codons
Transcription and Translation.
Gene expression
Site of active replication.
Replication fork
Initiation site of nucleic acid synthesis.
Promoter sequence
Triplets of nucleotide bases; Each codon codes for a specific single amino acid.
Codons
Anabolic process (Biosynthesis) vs Catabolic process (Biodegradation).
Metabolism
Amplified PCR product (Contamination in PCR lab).
Amplicon
Areas that are free from amplified products of PCR.
Clean room
Areas in which amplification is done (PCR room); may have contaminating amplicons.
Dirty room
Inactivates DNA in the sample leading to false negative PCR assay.
Dnase enzyme
Found ubiquitously in the environment and human body; can cause false negative PCR assay but can be inactivated by using RNAse-free materials or treatment with guanidinium isothiocyanate.
RNAse enzymes
Reduces background amplification in hybridization methods that could interfere in the analysis of relevant amplification signals.
Isocytidine (isoC) and isoguanosine (isoG)
Reduces carryover/contamination from PCR assays.
Uracil-N-Glycosylase (UNG)
Are number of cycles a significant rise of fluorescence (10x the SD) above background is detected. Also known as CT (Cycle threshold) or CP (Crossing point) or CQ (Cycle of Quantification).
CT value
The temperature in which DNA denatures/melts into two single strands.
Melting temperature (Tm)
These are a set of conditions that measure the likelihood of a double-stranded nucleic acid to dissociate into its constituent single strands.
Stringency
Single-stranded nucleic acid sequence that is used to identify a specific sequence of DNA/RNA from the sample with reporter molecule for detection of signals.
Probe
Single-stranded nucleic acid sequence that functions like a probe but without a reporter molecule. The primers can be specific for the genes that code for the microorganism’s genus, species, virulence factors, or antibiotic resistance.
Primer
Detection of a single nucleic acid target for detection and identification of a microorganism in a molecular assay.
Monoplex
Detection of multiple nucleic acid targets using cocktails of probes to detect multiple numbers of pathogens simultaneously in a single reaction. Disadvantage: Competition for resources among multiple amplifying sequences and cross-reaction between primers.
Multiplex
Fluorescence Resonance Energy Transfer, the transfer of energy from a donor dye molecule to an acceptor dye molecule.
FRET
Indications for molecular assays include hard-to-grow fastidious microorganisms, extensive delays in cultivation, lack of reliable methods for identification, and characterization of previously unknown organisms or strains of known organisms during epidemics, mutated organisms, or antibiotic resistance mechanisms. Organisms do not need to be alive.
Indications of Molecular Assays
Limitations of molecular assays include less sensitivity for multiplex assays, troublesome contamination, false positives, complex and technical methods, discriminatory properties, cost, and lack of standardization.
Limitations of Molecular Assays
Use plastic swabs rather than wooden or wired shafts. Do not use calcium alginate swabs with aluminum shafts as it interferes with amplification of nucleic acids. All materials including pipettes, surfaces, tubes, microcentrifuge tubes should be DNase/RNase-free to avoid degradation of sample nucleic acids leading to false-negative results.
Pre-analytical factors
Method for nucleic acid-based tests that involves the production and labeling of nucleic acid probes, preparation of target nucleic acid from the specimen, hybridization, and detection of hybridization.
Hybridization
Electrophoresis followed by staining with Ethidium bromide.
Southern Hybridization
A hybridization method that involves the detection of pathogen in the patient’s cell or tissues.
Fluorescence in situ Hybridization
Use of increased salt to lower stringency, which can result in double-stranded DNA becoming single-stranded, and increased temperature to increase stringency, often with the use of urea and formamide.
Stringency (Extraction)
Amplification steps
STEPS: 1. Extraction of Target nucleic acid from specimen 2. Amplification 3. Detection of amplified products.
DNA amplification or DNA xeroxy, invented by Kary Mullis, is the most common nucleic acid amplification method used in molecular diagnostics
POLYMERASE CHAIN REACTION (PCR)
Conventional PCR has —- repetitive thermal cycles: temperature-dependent
20-50
Extract nucleic acid with heat/chemical/enzyme.
Denaturation (94°C)
Primer (specific to target DNA sequence) is added to the denatured DNA.
Annealing (50-58°C)
Taq Polymerase is used. Can amplify a single copy into 10^7 to 10^8 copies.
Elongation (72°C)
A DNA or RNA segment extracted from the sample which serves as the target for PCR.
Template
Oligonucleotide that is specific for the target sequence present in the template.
Primers
Synthesizes new strands of DNA and must be thermostable for PCR.
DNA Polymerase
Also known as RNA-dependent DNA Polymerase, used ONLY in RT-PCR to synthesize complementary DNA (cDNA) from an RNA template.
Reverse Transcriptase
Cofactor of DNA Polymerase.
Magnesium Chloride (MgCl2)
Ensures proper pH for DNA Polymerase to function.
Buffer
Used by DNA polymerase to synthesize new DNA strands during the Elongation phase.
Deoxynucleotides (dNTPs)
Instrument used in PCR that adjusts temperature depending on the step being conducted.
Thermal Cycler
A heat-stable DNA polymerase used in PCR; taq polymerase.
Thermus aquaticus
A heat-stable DNA polymerase used in PCR, known for its proofreading ability; Pfu polymerase.
Pyrococcus furiosus
Heat-stable DNA polymerase used in PCR, with high fidelity (Wind or TLi polymerase or Vent polymerase)
Thermococcus litoralis
A heat-stable DNA polymerase used in PCR, capable of amplifying both DNA and RNA templates; Tth polymerase
Thermus thermophilus
Amplification followed by gel electrophoresis and band staining to detect amplified products.
Conventional PCR
PCR process where amplicon accumulation is detected in real-time by fluorophores that send fluorescent signals.
Real-time PCR (Quantitative PCR or qPCR)
Stages of Real-time PCR
Lag, Exponential, and Plateau phase
Real-time detection methods
5’Nuclease (TaqMan) PCR, SYBR Green, Dual-probe FRET, molecular beacon, Scorpion primers.
Real-time PCR commercial platforms
Roche LightCycler, Bio-Rad CFX96, 3M Integrated Cycler, GeneXpert (Cepheid), GeneAMP 5700, and Prism 7700.
Performed with two successive PCR reactions: 1st PCR amplifies a large fragment, 2nd PCR amplifies smaller fragments of the 1st PCR amplicons.
Nested PCR
Separates individual nucleic acids into ≥20,000 droplets using a microfluidic technique to analyze if each droplet has the target molecule.
Digital PCR
Uses RNA as the template; RNA is first converted into cDNA, then usual PCR steps proceed.
Reverse Transcription PCR (RT-PCR)
Real-time detection of amplicons made by amplifying RNA target sequences, especially for viruses (SARS-CoV-2, HIV, Hepatitis C, Ebola, Zika).
Real Time RT-PCR
Detection of multiple nucleic acid targets for one or more microorganisms in a single reaction.
Multiplex PCR
Inversely proportional to the nucleic acid concentration present in the sample.
CT value
Determines the exact nucleotide sequence of a single gene from an organism for identification and mutation analysis.
Nucleic acid sequencing
During amplification, pyrophosphate (PPi) is released and converted to ATP, generating light detected as a pyrogram.
Pyrosequencing
Identifying drug-resistant infections, bacteria, fungi, and viruses.
Pyrosequencing Uses
Receiving/Reception, Extraction, Reagent Prep, Template Addition, PCR, Data Management (Clean rooms; Dirty room with amplicons).
Sections of Molecular Laboratory
DNA molecules attached to a solid support, used to detect gene expression, mutations, and pathogen identification.
DNA Microarrays and Nanoarrays
Can detect gene expression and mutations or identify new genes, holding DNA of thousands of pathogens simultaneously.
DNA Microarrays
More sensitive than microarrays, capable of screening more molecules without coupled reporters.
Nanoarrays
Rapid identification and antimicrobial susceptibility testing; Identification within 2 hours using cartridge and FISH probes for bacteria and fungi; MIC determination within 7 hours using morphokinetic cellular analysis via dark-field microscopy; Same day AST results; FDA approved for blood culture, others in progress.
Accelerate Pheno System
By Giles Scientific USA; Utilizes digital imaging to automate zone of inhibitions reading, colony counting, and examination of chromogenic agar.
TRINITY V3
By Giles Scientific USA; Utilizes digital imaging to automate CLSI/EUCAST interpretations for broth microdilution, agar dilution, disk diffusion, MIC strip/E-test, organism identification through API, RapID, or Liofilchem, colony count, and urine screening.
BIOMIC V3
