Laboratory Techniques Flashcards
Southern Blot
- DNA sample is enzymatic ally cleaved into smaller pieces, which are separated on a gel by electrophoresis, and then transferred to a filter
- Filter is exposed to radiolabeled DNA probe that recognizes and annuals to its complimentary strand
- Resulting DS, labeled piece of DNA is visualized when filter is exposed to film
Northern Blot
RNA sample is electrophoresis
Useful for studying mRNA levels, which are reflective of gene expression
Western blot
Simple protein is separated via gel electrophoresis and transferred to a membrane
Labeled Ab is used to bind to relevant protein
e.g. Confirmatory test for HIV positive ELISA
Southwestern blot
Identifies DNA-binding proteins (e.g. Transcription factors) using labeled oligonucleotide probes
Flow cytometry: mechanism
Lab technique to assess size, granularity and protein expression (immunophenotype) of individual cells in a sample
Cells are tagged with Ab specific to surface or intracellular proteins. Ab then tagged with a unique fluorescent dye. Sample is analyzed one cell at a time by focusing a laser on the cell and measuring light scatter and intensity of fluorescence
Flow Cytometry: use
Data are plotted either as histogram or scatter plot
Commonly used in work up of hematologic abnormalities (e.g. Paroxysmal nocturnal hemoglobinuria, fetal RBCs in mothers blood and immunodeficiencies - CD4 count in HIV)
Microarrays: Mechanism
Thousands of nucleic acid sequences are arranged in grids on glass or silicon.
DNA or RNA probes are hybridized to the chip and a scanner detects the relative amounts of complementary binding
Microarrays: Use
Used to profile gene expression levels of thousands of genes simultaneously to study certain diseases and treatments
Able to detect single nucleotide polymorphisms and copy number variations for a variety of applications including genotype get, clinical genetic testing, forensic analysis, cancer mutations, and genetic linkage analysis
Enzyme-linked immunosorbent assay (ELISA)
Immunologic test used to detect the presence of either a specific Ag (e.g. HBsAg) or Ab (e.g. Anti-HBs) in a pt’s blood sample.
Detection involves the use of an Ab linked to an enzyme. Added substrate reacts with enzyme producing a detectable signal (e.g. Color change)
Major ELISA variations include direct, sandwich, and competitive
Karyotyping
A process in which metaphase chromosomes are stained, ordered, and numbered according to morphology, size, arm-length ratio, and banding pattern
Can be performed on a sample of blood, BM, amniotic fluid or placental tissue
Used to diagnose chromosomal imbalances (e.g. Trios is, sex chromosome disorders)
Fluorescence in situ hybridization (FISH)
Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes
Used for specific localization of genes and direct visualization of chromosomal anomalies at the molecular level
FISH: microdeletion
No fluorescence on a chromosome compared to fluorescence at the same locus on the second copy of that chromosome
FISH: Translocation
Fluorescence outside the original chromosome
FISH: Duplication
Extra site of fluorescence on one chromosome relative to its homologous chromosome
Cloning methods
Cloning is the production of a recombinant DNA molecular that is self-perpetuating
Steps:
1. Isolate eukaryotic mRNA (post RNA processing steps) of interest
2. Expose mRNA to RTase to produce cDNA (lacks introns)
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes
4. Transform recombinant plasmid into bacteria
5. Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA)
