Laboratory Techniques Flashcards
Best test to determine whether a gene is being expressed
Northern blot
What is the polymerase chain reaction used for
Amplify a desired fragment of DNA
Polymerase chain reaction steps
- Denaturation (DNA is denatured by heating to separate the strands)
- Annealing (during cooling, excess DNA primers anneal to a specific sequence on each strand to be amplified)
- Elongation (heat-stable DNA polymerase replicates the DNA sequence following each primer)
What is RT-PCR used for
It is used to detect and quantify levels of messenger RNA in a sample
*mRNA - RT - cDNA - PCR - amplification
Southern blotting is used to measure what type of molecule
DNA
Enzymes used in southern blotting to cleave DNA samples in smaller pieces
Restriction endonucleases
*Restriction site mutations can be detected using this because they alter DNA lengths
Blotting technique used to assess level of gene expression
Northern blot
*As it studies mRNA levels, these are reflective of gene expression
Blotting technique that uses labeled antibodies to bind relevant proteins
Western blot
Blotting technique that identifies DNA-binding proteins using labeled oligonucleotide probes (dsDNA)
Southwestern blot
Cellular characteristics evaluated by flow cytometry
Size, granularity, and protein expression (immunophenotype)
Describe the process of flow cytometry
Cells are tagged with antibodies specific to surface or intracellular proteins - these are then tagged with a unique fluorescent dye - sample is the analyzed one cell at a time by focusing a laser on the cell and msasuring light scatter and intensity of fluorescence
Laboratory technique used to profile gene expression levels of thousands of genes simultaneously
Microarrays
Laboratory technique that is able to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs)
Microarrays
Laboratory technique that detects specific antigens or antibodies through the use of a reaction with an antibody-linked enzyme
Enzyme-linked immunosorbent assay
To do a karyotype, chromosomes need to be in which cell cycle phase
Metaphase
Laboratory technique that uses fluorescent DNA or RNA probes that bind to specific gene sites of interest on chromosomes
Fluorescence in situ hybridization (FISH)
Main difference between chromosomal DNA and complementary DNA
cDNA lacks introns
Laboratory technique used to manipulate genes at specific developmental points (eg, study a gene whose deletion causes embryonic death)
Cre-lox system
How does RNA interference down-regulates gene expression
dsRNA is synthesized that is complementary to the mRNA sequence of interest - when transfected into human cells, dsRNA separates and promotes degradation of target mRNA
