lab pcr electroporesis
DESCRIPTION
PCR electrophoresisTRANSCRIPT
LABORATORY ASPECT OF PCR, IMMUNOASSAY ELECTROPHORESIS
Dr.Budi Dermawan Lubis, SpPK
Protein Electrophoresis Electrophoresis separates protein based on their electric
charge densities.
At pH > isoelectric point protein negatively charged and moved to anode on agarose or cellulose acetate gel.
Sample: serum, urine, CSF, EDTA for Hb electrophoresis
Protein Electrophoresis
Absolute protein fraction: percentage x TTP
Caution: plasma contain fibrinogen can be mistake as paraprotein, intravascular hemolyticà Hb-haptoglobin à ↑ α2 band, light chain myeloma à no M spike detected on SPE
Immunoassay • Sample collection and processing is a complex à
no simple system that will meet the requirements for all analyte, it is important to determine the type of specimen required before begins to obtains the specimen
• Immmunoassay : 1ml blood for 3- 4 tests • Haemolysis : in vivo or in vitro, >300mg/dl à red color of serum, increase intracellular constituents (K, LDH), decrease
extracellular constituent, interference with analytical assay • Lipaemic : caused by elevated lipoprotein , plasma turbidity at TG concentrations > 300 mg/dl, interference with photometric & electrophoretic analysis • Icteric : Interference with spectral & chemical (oxidase / peroxidase • Centrifuge sample 1200g 10minute for kompleks AG-AB with turbidimetry method
Laboratory Criteria For Unacceptable Samples
1. Inadequate sample identification
2. Inadequate volume of blood collected into an
additive tube
3. Use of an improper collection tube
4. Hemolysis
5. Improper transportation & storage : sample for
blood gases transported on ice
6. Contamination suspected
7. Unknown time delay
Immunoassay
Polymerase Chain Reaction
• Rapid and highly-specific amplification of DNA fragments
• Components underlie the PCR process:thermostable DNA polymerases and the ability to specific oligonucleotide primers
• PCR consists of three stages, multiply repeated: denaturing, annealing, and elongation. After the reaction is completed amplicon can be visualized by gel electrophoresis.
• Advantage: rapid, sensitive, specific, robust. Disadvantage: expensive, extraneous DNA may contaminate reaction, mutation gen have to design primer.
• Indication : Dx malignancy, bacteria, viruses, thallasemia.
Temp Time Σ Cycle
Denaturation Annealing Elongation
94°C 50-65°C
72°C
15-30 s 30-60 s
45 s – 3 min
25 - 30
PCR