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Lab Tech
First Aid - Biochem - Lab Tech
| Question | Answer |
|---|---|
| PCR - basics | amplifies a desired fragment of DNA, 1.denaturation, 2.annealing, 3.elongation > these steps repeated multiple times for DNA sequence amplification *agarose gel electrophoresis = size separation of PCR products (smaller molecules travel further) |
| PCR - 1.denaturation | DNA is denatured by heating to generate 2 separate strands |
| PCR - 2.annealing | during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplified |
| PCR - 3.elongation | heat-stable DNA polymerase replicates the DNA sequence following each primer |
| Blotting procedures - SNoW DRoP | Southern = DNA Northern = RNA Western = protein |
| Southern blot | DNA sample electrophoresed on a gel and then transferred to a filter - soaked in denaturant and exposed to labeled DNA probe that recognizes and anneals to complementary strand - double stranded labeled piece of DNA is visualized filter is expo to film |
| Northern blot | similar to Southern except that Northern blotting involves radioactive DNA probe binding to sample RNA |
| Western blot | sample protein is separated via gel electrophoresis and transferred to a filter, labeled antibody is used to bind to relevant protein |
| Microarrays | thousands of nucleic acid sequences are arranged in grids on glass or silicon - DNA or RNA probes hybridized to chip, scanner detects relative amounts of complementary binding, used to profile gene expression levels or to detect SNP |
| ELISA (enzyme-linked immunosorbent assay) | -rapid immunologic tests for antigen-antibody reactivity - often used for HIV testing to find anti-HIV, sensitivity and specificity of ELISA approach 100% but both false positives and negatives do occur |
| ELISA method | 1.test antigen (coupled to color generating enzyme CGE)to see if recognized by immune sys. or 2.test antibody (with CGE)to see if a certain antigen is present - if target is present test solution will have an intense color reaction = positive test result |
| FISH (fluorescence in situ hybridation) | fluorescent DNA or RNA probe binds specific gene at site of interest - used for specific localization of genes, direct visualization of anomalies(microdeletions) at molecular level (when deletions are too small to be seen by karyotype) *fluorescene-gene + |
| Sanger DNA sequencing | dideoxynucleotides halt DNA polymerization at each base, creating sequences of various lengths that encompass entire original sequence - terminated fragments are electrophoresed and the original sequence can be deduced |
| Karyotyping | metaphase chromosomes are stained, ordered, and numbered based on shape, size, banding, performed on blood, bone marrow, aminotic fluid, placenta - dx. chromosome imbalances (trisomies, deletions, sex chromosome disorders) |
Created by:
Smukadam
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