Zaidi: Analyzing Cell, Molecules and Systems 2 Flashcards
Restriction endonuclease
Enzymes isolated from bacteria
Cut DNA at specific sites
cDNA
No introns
Requires reverse transcriptase
PCR purpose
Earlier detection of microorganisms: HIV, bacterial/fungal infections
Detection of specific genetic mutations
PCR technique
Heat double stranded DNA to denature
Introduce primers that flank each end of DNA in 3->5
Cool and allow primers to anneal
Add deoxy nucleotide triphosphates dNTPs
Taq polymerase
Synthesizes copy of DNA by extending the primers on both ends. DNA doubles in each cycle and becomes amplified
Advantage of PCR
Very small amount of template DNA needed
10^9 fold amplification
Disadvantage of PCR
Need to know the sequence of the flanking DNA for primer design
Error prone
Amplification of contaminating DNA
Quantitative PCR used to
Quantify copy number of a specific gene in two or more samples in real time
qPCR uses what in addition to primers
A probe which fluoresces only in presence of PCR product
-usually a complementary oligo with a fluorescent tag
qPCR uses
Detect levels of an infectious agent
Determine levels of gene expression
Restriction fragment length polymorphism
Individual genomes differ by 1 in every 1000 base pairs
Some of these occur in the recognition sequences for restriction enzymes
When two genomes are cleaved with a restriction enzyme, the pattern is different
RFLP uses
DNA fingerprinting
Used in forensic analysis, paternity testing, disease detection
Variable number of tandem repeats VNTR
Pattern of short tandem repeats occurs in genome but varies in individuals
Useful in identification and severity of inherited diseases
Huntingtons, fragile x, frederich ataxia
DNA microarrays
Used to determine overall change in gene expression between two samples
