Biochemistry-Lab Techniques Flashcards
Describe PCR
test used to amplify a desired fragment of DNA. The steps include:
- Denaturation- DNA is denatured by heating to generate 2 seperate strands
- Annealing- during cooling, excess premade 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
These steps are repeated multiple times for DNA amplification
What is agarose gel electrophoresis?
used for size seperation of PCR products (smaller travels further); compared to a DNA ladder
What is a Southern Blot?
A DNA sample is enzymatically cleaved into smaller pieces, electrophoreses on a gel, and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a radiolabeled DNA probe that recognizes and anneals to its complementary strand.
The resultant double-stranded, labeled piece of DNA is visualized when the filter is exposed to film
What is a Northern Blot?
Similar to a southern blot, except that RNA is processed. Useful for studying mRNA levels, which are reflective of gene expression
What is a Western Blot?
Sample protein is seperated via gel electrophoresis and transferred to a filter, A labeled Ab is used to bind to relevant protein. Confirmatory test for HIV after ELISA
What is a Southwestern Blot?
ID’s DNA-binding proteins (e.g. transcription factors) using a labeled oligonucleotide probe
What are microarrays?
Thousands of nucleic acid sequences are arranged in grids on silicon. DNA or RNA probes are hybridized to the chip and a scanner detects the relative amounts of complementary binding.
Used to profile gene expression levels of thousands of genes simultaneously to study certain disease,
Able to detect SNPs and copy number variations
Describe an ELISA
Used to detect the presence of either a specific antigen or a specific Ab in a blood sample
The pt’s blood is probed with either:
Direct ELISA: uses a test Ab to see if a specific antigen is present. The Ab is directly coupled to a color-generating enzyme to detect the antigen
Indirect ELISA: uses either a test antigen or AB to see if a specific Ab or antigen is present. A secondary Ab coupled to a color-generating enzyme is added
What is 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 blood sample, bone marrow, amniotic fluid, or placental tissue.
Used to diagnose chromosomal imbalances
What is Fluorescence in situ hybridization?
Fluorescent DNA or RNA probe binds to specific gene site of interest on chromsomes. Used for specific localization of genes and direct visualization of anomalies (e.g. microdeletions) at the molecular level (when detection is too small for karyotyping)
Fluorescent= gene is present
How is cloning performed?
Cloning is the production of a recombinant DNA that is self-perpetuating
- Isolate eukaryotic mRNA of interest
- Expose mRNA to reverse transcriptase to produce cDNA (lacks introns)
- Insert cDNA fragments into bacterial plasmids containing Ab resistance genes
- Transform recombinant plasmid into bacteria
- Surviving bacteria on antibiotic medium produce cloned DNA (copies of cDNA)
What is a Cre-lox system?
can inducibly manipulate genes at specific developmental points (e.g. to study a gene whose deletion causes embryonic death)
What is a RNA interference?
dsRNA is synthesized that is complementary to the mRNA sequence of interest. When transfected into human cells, dsRNA seperates and promotes degradation of target mRNA, “knocking down” gene expression
