Lab techniques - DONE Flashcards
PCR
used to amplify desired grament of DNA
usedfool as a diagnostic tool
Steps:
denaturation - DNA denatures by heating and generating 2 separate strands
annealing- during cooling, excess premade DNA primers anneal to a specific sequence on each strand to be amplfied
elongation- heat-stable DNA polymerases replicate DNA sequence following each primaer
these steps are repeated multiple times for DNA sequence amplification
agarose gel electrophoresis - used for size separation fo PCR products and compared against DNA ladder
Blotting procedures
Southern blot
Northern Blot
Western Blot
Southwestern Blot
Southern Blot
DNa sample enzymatically cleaved into smaller pieces
electrophorese on a gel and transferred onto a filter
filter socked in a denaturant and sunsequently exposed to a radiolabeled DNA probe that recognizes and aneals to its complementary strand
results in DB stranded, labeled pieces of DNA that is visualized when filter is exposed to film
mnenomic for blots
SNoW DRoP:
Southern = DNA
Northern= RNA
Western= Protein
Northern Blot
Similar to southern except RNA is used
useful for studying mRNA levels which is indicativ of gene expression
Western Blot
sample protein separated via gel elecrophoresis
transferred to filter
labeled Abs used to bin to relevant protein
confirmatory test for HIV after (+) ELISA
Southwestern Blot
Identifies **DNA binding proteins **= ex: transcriptional factors - using labeled oligonucleotide probes
Microarrays
1000s of nucleic acid sequences arranfed in grids on glass or silicon
DNA/RNA probes hybridized to chip
scanner detects releative amt of complementary binding
used to profil gene expression levels of 1000s of genes simulatenously to study certain diseases and tratments
Able to detect single nucleotide polymorphisms ( SNPs) and copy number variations ( CNVs) for genotypes/clinical genetic testing/forensive analysis/etc
ELISA
used to detect presence of either a specific antigen ( direct) or specific Ab ( indirect) in a pt’s blood sample
it target substance is present, test solution will change color indicating (+) test result
both sensitivity and specificity of ELISA appraches 100% but false + and - is possible
Indirect ELISA
using test antigen to see if a specific Ab is present in blood
a secondary Ab is coupled to a color-generating enzyme and added to detect first Ab
Direct ELISA
use a test Ab to see is a specific Ag is present in blood
secondary Ab couple with color generating enzyme added to detect Ag
FISH
( flourescence in situ hybridization)
flourescent DNA or RNA bind specific gene site of interest on chromo
used for specific localization of genes and direct visualization of anomalies - ex: microdeletions - at the molectural level
flourescence = gene is present
no flourescence = gene has been deleted
Cloning methods
cloning is the production of recombinant DNA moleculte that is self-perpetuating
steps:
isolate eukaryotic mRNA of interest
expose mRNA to reverse transcriptase to produce cDNA ( lacks introns)
insert cDNA fragment into bacterial plasmids contaning antibiotic resistance genes
transform recombinant plasmid into bacter
surviving bacteria on AB medium produce cDNA
Gene expression modification
transgenic strategies in mice invole:
- random insertion of gene into mouse genome
- targeted insertion/deletion of gene through homo recomb with mouse gene
Cre-lox system: manipulate geners at specific developmental points
RNA interference
dsRNA synthesized that is complementary to mRNA sequence of interest
when transfected into human cells, dsRNA separates and promotes degradation of target mRNA , ‘knocking down’ gene expression
