Oligonucleotide labelled with a radioactive/fluorescent tag
Name 3 nucleic acid amplification techniques
PCR Nucleic acid sequence-based amplification transcription mediated amplification strand displacement amplification
What goes into a PCR reaction?
Purified nucleic acid from the sample of interest Primers Taq polymerase Mg2+ Tris Ph 8.4 dNTPs
Describe the steps in a PCR reaction
Denaturation: Reaction heated to 95C for 30s to activate Taq polymerase and denature the DNA (melting of the template) which produces single-stranded DNA. (Annealing temperature is dependent on the GC content) Annealing: Reaction temperature is lowered to 50-65C for 20-40s to allow primers to anneal to the DNA template. Extension: Reaction heated to 75C which is the optimum temperature for Taq polymerase. Taq synthesises new strands of DNA complementary to the template by adding dNTPs Cycle is repeated 30-40 times to amplify the DNA.
How are PCR products analysed?
Gel electrophoresis Southern blot
Give 3 disadvantages of standard PCR
Gel electrophoresis has limited specificity End point analysis is not quantitative Analysis of PCR products can result in contamination of the lab
How is DNA sequenced?
dideoxy sequencing. Reaction mixture includes standard dNTPs and dideoxydNTPs which are labelled with a fluorescent dye. dideoxydNTPs are chain terminating. Random incorporation of ddNTPs produces a series of DNA fragments of variable length. Separation of the fragments by size produces a sequencing ladder as a series of coloured bands which are read by software.
What are the benefits of RT-PCR?
Amplifies and detects the target DNA using fluorogenic probes to monitor the amplification process. Allows quantification of input DNA Accurate Analysis performed on computer (highly automated) without opening tube, reduces contamination risk Can be used for diagnosis and monitoring of infectious disease
What goes into an RT-PCR reaction?
Nucleic acid from sample Taq polymerase Primers Probe dNTPs Mg2+ Tris pH8.4
Describe how melting curve analysis is used
The temperature DNA strands melt (separate) at when heated can vary depending on the sequence, length and GC content. The temperature at which 50% of the DNA is denatured is the melting point.
Single base differences in heterozygous DNA can change its melting profile. Therefore can be used to identify and genotype DNA products.
The temperature of the reaction mixture is increased steadily. Probes dissociate from the target DNa at specific temperatures. A point mutation causes a mismatch between the target and probe, so the probe melts at a lower temperature.
How are products of RT-PCR quantified?
The number of cycles required for the fluorescent signal to reach threshold are measured.
These values are then compared to a calibration curve with a range of known standards. A standard plot is drawn to show cycle time and starting quantity. Sample is plotted against graph to calculate starting quantity.
Name 4 uses of PCR
Viral load monitoring
Mutation detection (viruses, genomic DNA)
Detection of specific mutations (e.g. cancers)
Detection of mRNA
When would you use serology for virally infected patients?
Immunity screen (resposne to vaccine, detection of previous infection)
Chronic infections (HIV, HCV)
Clearance of recent infection (e,g. VZV IgM)
When would you use a nucleic acid amplification test for virus detection?
Acute infections (respiratory viruses)
Immunosuppressed patients (CMV, EBV infection after transplantation, HCV infection)
Neonatal infection (maternal Igs can be misleading, baby not developed Igs yet).
What are the signs and symptoms of a wound infection?
Redness or excessive swelling in the wound area
Throbbing pain or tenderness in the wound area
Red streaks in the skin around the wound/progressing away from the wound
Pus/watery discharge deneath the skin or draining from the wound
Tender lumps/swelling in axillary, inguinal are cervical modes
Foul odour from the wound.
What things should you assess in a patient with a suspected infection?
Source: will help idenfity possible causative organism and where sample should be taken from
Immunity: (is the patient immunocompomised?) Will ahve a bearing on the range of pathogens.
Travel: has the patient been exposed to unusual organisms
Growth of organisms in tissues that produces an associated host reaction
Presence of organisms in tissues with no host response
How is drug sensitivity of an organism determined?
Disk diffusion of antibiotics.
Agar plate is seeded with the isolate of interest. Pre-prepared discs containing a standard concentration antibiotic are evenly dispensed onto the agar surface. The antibiotic begins to diffuse outward from the discs creating a gradient.
The plate is incubated overnight and growth is observed. If the bacteria is suscptible there will be a zone of inhibition around that disc.