Laboratory techniques Flashcards
What is and how is PCR done?
Amplifies a fragment of DNA, it’s useful as diagnostic tool. (Neonatal HIV, herpes encephalitis…)
- Denaturation: DNA heated to 95ºC to separate strands.
- Annealing: cooled to 55ºC. Addition of DNA primers, heat-stable DNA polymerase (Taq) and deoxynucleotide triphosphates (dNTP). DNA primers anneal to specific sequence, amplifies on each strand.
- Elongation: Temperature goes to 72ºC, DNA polymerase attaches dNTP to the strand to replicate sequence after each primer.
Heating and cooling cycles until DNA sample size is sufficient.
What is Reverse transcriptase polymerase chain reaction?
Detects and quantifies mRNA levels.
It uses reverse transcription to create a complimentary DNA template that is amplified via standard PCR.
What is CRISPR/Cas9?
Genome editing tool derived from bacteria.
It is a guide RNA (gRNA) complementary to a target DNA sequence and an endonuclease (Cas9) that makes single or double strand break at target site.
Imperfectly repaired break by nonhomologous end joining (NHEJ): accidental frameshift mutations (knock out) or a donor DNA sequence can be added to fill the gap, using homology directed repair (HDR).
Might be used to remove virulence factors, replace disease-causing alleles and targeting tumor cells.
Which are the blotting procedures?
Southern, northern, western and southwestern.
What is southern blot?
DNA sample is enzymatically cleaved, pieces are separated on gel by electrophoresis and transferred to a filter which is then exposed to radiolabeled DNA probe that recognizes and anneals to its complimentary strand, the resulting double-stranded labeled piece of DNA is visualized when filter is exposed to film.
What is northern blot?
Like southern, but RNA sample is electrophoresed instead of DNA. Useful for studying mRNA, reflective of gene expression.
What is western blot?
Sample protein is separated via gel electrophoresis and transferred to a membrane, labeled antibody is used to bind relevant protein.
What is flow cytometry?
Asses size, granularity and protein expression immunophenotype of individual cells in a sample.
Used in workup of hematologic abnormalities (leukemia, paroxysmal nocturnal hemoglobinuria, fetal RBC in mother blood) and immunodeficiencies.
How does flow cytometry work?
Cells are tagged with antibodies to surface of intracellular proteins. The Abs are tagged with a fluorescent dye and sample is analyzed by focusing on the cell and measuring light scatter and intensity of fluorescence.
Data is reported as a histogram or scatter plot in two measures.
What are microarrays?
Nucleic acid sequences are arranged in grids on glass or silicon, the DNA/RNA probes are hybridized to the chip and a scanner detects relative amounts of complementary binding.
Profile gene expression of thousands of genes simultaneously to study diseases and treatments.
Genotyping, clinical genetic testing, forensic analysis, cancer mutations and genetic linkage analysis.
What is enzyme-linked immunosorbent assay?
Detects specific antigens or antibodies, it involves the use of antibodies linked to an enzyme, the added substrate reacts producing a detectable signal. High s/s but less specific than western blot.
What is karyotyping?
Colchicine to stop chromosomes in metaphase, they’re stained, ordered and numbered (morphology, size, arm-lenght ratio and banding pattern).
Blood, bone marrow, amniotic fluid, placental tissue.
Dx chromosomal imbalances.
What is and how does FISH work?
The fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes. It’s used for localization of genes and direct visualization of chromosomal abnormalities at molecular level.
Microdeletions, translocation, duplication.
What is molecular cloning?
The production of a recombinant DNA molecule in a bacterial host.
- Isolate euka mRNA of interest
- Add reverse transcriptase to produce complimentary DNA
- Insert cDNA fragmentsinto bacterial plasmids containing antibiotic resistance genes
- Insert recombinant plasmid into bacteria
- Surviving bacteria on antibiotic medium produce cloned DNA
What are the gene expression modifications?
Random insertion (constitutive expression) of gene into genome, targeted insertion (conditional expression) or deletion of gene through homologous recombination with host gene. Knock-OUT=removing a gene. Knock-IN=inserting a gene.
