Molecular Diagnostic Techniques Flashcards
Sensitivity
True + rate
Specificity
True negative rate
Electrophoresis
Separates negatively charged molecules, moves to positive end
What size and structure of molecules move faster in electrophoresis?
- The smaller the faster
- Supercoiled > linear > nicked circles (DNA)
Restriction fragment length polymorphism (RFLP)
Analyzes differences in homologous DNA seq. by restriction enzymes
used in forensics and parenthood testing
Restriction enzymes
cut DNA at specific recognition nucleotide sequences
Sequence specific!
Ex:
EcoRI- sticky end
Smal- blunt end
Example of Restriction enzyme technique?
Cryptosporidium spp. infections
Types of hybridization
- In situ
- Southern
- Northern
- Microarray
What is hybridization?
Denatured, single stranded DNA
PROBE binds to coplementary single stranded sequence
Probe
Hybridization
- High SPECIFICITY
- labeled using radioisotope, enzyme, or chemiluminescence
- detects complementary sequences
- varies in size
PCR primer
single stranded DNA fragments, determines SPECIFICITY
distance between primer binding sites determines size of PCR product
random or specific
Real Life/ Quantitative PCR
- probe or dye to generate a fluorescent signal
- Signal in REAL TIME allows quantification of starting material
- Lag phase inversely proportional to the amount of starting material
lag phase, log phase, stationary phase
Isothermal amplification (LAMP)
-Quick! 1 hr
- greater sensitivity than PCR
- Visible results
-No need for temperature cycling
Cons: Primer set complicate
Nucleic acid amplification
Target amplification: enzyme mediated process to make copies of targeted nucleic acid
PCR testing
Highly sensitive, false positives
What do primers determine?
- Product size
- Annealing temperature
- Specificity
- Nucleotide composition
