MD Exam 2 Flashcards
Detection system in MD should have three qualities
Sensitivity, specificity, simplicity
Sensitivity
the test must be able to detect very small amounts of target even in the presence of other molecules
Specificity:
the test yields a positive result for the target molecule only
Simplicity:
the test must be able to run efficiently and inexpensively on a routine basis
Regulation Organization and Agents
CLIA
FDA
CMS
CAP
JCAHO
COLA
Clinical validation: Accuracy
a
Clinical validation: Precision
a
Clinical validation: analytic sensitivity
a
Clinical validation: Analytical specificity
a
Clinical validation: Diagnostic specificity
What do we use to lyze cells for NA isolation and analysis?
Detergent SDS, Tween 20
What methods do we use for assessment of quality and quantity?
UV Spectrophotometry (O.D 260-280) and fluorescent dyes
OD260=
1.0 ~50 ug/ml of dsDNA or 4- ug/mL of RNA or ssDNA
After you isolate the DNA you can do direct detection of NA w/
- Restriction endonuclease enzyme digestion
- Gel electrophoresis and ETBr Stain
- Restriction Fragment Length Polymorphism (RFLP)
Restriction Endonucleases (RE) are found only in
microorganisms
RE’s are homodimers that recognize
symmetric dsDNA (palindromes)
Palindromes:
Read the same in both directions
Recognize specific sequences of 4, 5, or 6 nucleotides
Cut by breaking phosphodiester bonds in both strands
Cut DNA into smaller pieces
Gel electrophoresis separates molecules on the basis of __________ by:
their charge and size; retard the passage of molecules according to their size and shape
How do (-) and (+) particles move in gel electro
The negatively charged particles move toward the positive electrode while the positive charged particles move toward the negative electrode
What are the two types of gels?
Agarose and acrylamide
HB^S produces
sickled cell RBC
Homozygotes for HBs are anemic. What is the lost site?
Mst II
Blotting/Hybridization Techniques uses
specific probes that are labelled specific sequences of DNA can be identified
To recognize RNA, which blotting method do we use?
Northern blot (using DNA or RNA probes)
To recognize DNA, which blotting method do we use?
Southern (using DNA or RNA probes)
To recognize protein, which blotting method do we use?
Western (antibody)
Probes are
ssDNA that will base pair with a complementary sequence of either RNA or DNA
ID of target detected/identified by labeling probes with:
Radioactive (P32) label
Chemiluminescent compound
Fluorescent compound
Enzymatic label (alkaline phosphatase/horseradish peroxidase with substrates to get color reaction done)
ID of target detected/identified by labeling probes with:
Radioactive (P32) label
Chemiluminescent compound
Fluorescent compound
Enzymatic label (alkaline phosphatase/horseradish peroxidase with substrates to get color reaction done)
Allele Specific Oligonucleotide Probes (ASO’s) must have
2 probes (1 for each allele)
Allele Specific Oligonucleotide Probes (ASO’s) must have
2 probes (1 for each allele; 1 normal, one mutant)
ASO is used for detection of Cystic fibrosis with what two probes?
AsOn = WT
ASOx = mutant cystic fibrosis gene (loss of phenylalanine 508)
Hybrid capture assays uses antibody capture assay to detect/capture
hybrid (DNA/RNA) molecules
Hybrid capture assay i used to detect
HPV
Each PCR cycle increases the number of strand by multiples of
2 or 2^n
Taq polymerase is isolated from bacterium
T. aquaticus
RT-PCR is done for
RNA viruses to convert into DNA
NASBA and TMA are more used in clinical settings because
they belong to isothermal PCR and they begin with RNA molecules
Signal amplification is not a target PCR, it targets
signals with bDNA branched DNA probes
Signal amplification quantifies
HIV-1, HCV RNA
Gold standard of molecular?
DNA sequencing
Sanger sequencing requires
ssDNA template, DNA primer, DNA polymerase, labeled nucleotides and modified nucleotides to terminate DNA elongation
ddNTPs will
prevent addition of further nucleotides
FISH is used for cytogenetics by detecting
large fragment of DNA and chromosomal abnormalities
