(5) Molecular Diagnostics Flashcards
What are two techniques used to detect infectious agents and diagnose inherited disorders?
- Hybridization
- PCR
Hybridization:
Describe
Single stranded DNA binds to another strand of DNA or RNA with complementary sequence to form:
DNA-DNA hybrid or DNA-RNA hybrid
Hybridization:
useful for?
Detection and quantification of target DNA or RNA
Hybridization:
What is a probe?
Short, single-stranded oligonucleotide
Hybridization:
What do you do with Target DNA?
Converted to single stranded DNA then IMMOBILIZED on a solid support; called blotting
Hybridization:
What is southern blotting?
Probe=DNA
Target=DNA
Hybridization:
What is northern blotting?
Probe=Single stranded DNA
Target=mRNA
Hybridization:
What do probes have on them?
Labeled with radioactive or fluorescent tag
Blotting Techniques:
Purpose?
Detection and visualization of specific biomolecules
Blotting Techniques:
Southern: Target?
DNA
Blotting Techniques:
Southern: Purpose?
Determine which restriction fragments are associated with a gene
Blotting Techniques:
Northern: Target?
RNA
Blotting Techniques:
Northern: Purpose?
Measure size and quantities of mRNA molecule
Blotting Techniques:
Western: Target?
Protein
Blotting Techniques:
Western: Purpose?
Measures amount of protein or antibody
Blotting Techniques:
Eastern: Target
PTM (lipid, carb, phosphorylation)
Blotting Techniques:
Eastern: Purpose?
Detects post-translational modifications (PTMs) on proteins
Polymerase Chain Reaction (PCR)
Describe
DNA subjected to high temp to denature
Primers complement sequences to flank each end of DNA
Allowed to anneal
Add 4 dNTPs
Taq Polymerase; synthesizes copy of DNA by extending the primers on both ends
Polymerase Chain Reaction (PCR)
Advantage?
Very small amount of template DNA needed!
10^9 fold amplification from trace amount of DNA
Polymerase Chain Reaction (PCR)
Disadvantage?
Need to know the sequence of the flanking DNA for primer design, error prone, amplification of contaminating DNA
Polymerase Chain Reaction (PCR)
What are the 3 major steps?1
- Heat to separate
- Cool to anneal
- DNA synthesis
Quantitative PCR (qPCR)
Used for?
Quantifying copy number of a specific gene in two or more samples in real time
- Detect levels of infectious agent
- Determine levels of gene expression
Quantitative PCR (qPCR)
How does it differ from traditional PCR?
In addition to primers, includes…
[a probe that fluoresces only in presence of the PCR product]
Detections of Variations in DNA sequences:
2 methods?
- Restriction Fragment Length Polymorphism (RFLP)
2. Variable number of tandem repeats (VNTR)
How can determining variations in DNA sequences be useful?
- Forensics
- Diagnostic (amniocentesis, newborn screening, genetic carriers)
RFLP:
Thought process behind?
- Genomes differ by 1 in every 1000 base pairs
* Some of these differences occur in the RECOGNITION SEQUENCES for restriction enzymes
RFLP:
How are RFLP used?
- DNA fingerprinting
- Forensic analysis
- Paternity testing
- Disease detection
RFLP:
Example of variation in restriction sites?
-Normal Beta-globulin allele has 3 restriction sites
vs
-Sickle cell only have 2 restriction sites
What does VNTR stand for?
Variable number of tandem repeats
VNTR:
Thought process behind?
-Pattern of short tandem repeats (STR) occurs in genome but varies in individuals
VNTR:
Useful for?
Identification and severity of inherited diseases
ex: Huntington disease
What process do you use to diagnose Huntington’s disease?
VNTR
What are examples RECOMBINANT PROTEINS that are produced on a large scale?
- Insulin
- Growth hormone
- Erythropoietin
- Clotting factors
- Vaccines against diseases such as flu and malaria
How are recombinant proteins formed?
cDNA of the protein inserted into expression vectors
How does human synthesized insulin differ from artificial?
Human produced insulin: Pro at position 28, Lys at pos 29 at C terminus of B chain
Lispro: reverse position of these 2 aa
Insulin aspart: proline 28 replaced by aspartic acid
What are the two artificial insulins?
Lispro, Insulin aspart
What are the benefits of Lispro and Insulin aspart?
Faster acting, more readily absorbed
What is ELISA?
Enzyme-linked immunosorbent assay (ELISA)
-Immunological technique which tests for levels of specific antigen or antibody concentrations
What does:
INDIRECT ELISA measure?
Amount of an ANTIBODY in a sample
What does:
SANDWICH ELISA measure?
Amount of an ANTIGEN in a sample
What technique is used to diagnose HIV?
**Indirect ELISA
must confirm w/ Western Blotting
What technique is used to detect an MI?
ELISA;
Cardiac forms of T and I increase in acute myocardial infarction
***Sandwich ELISA
What technique is used for pregnancy tests?
*** Sandwich ELISA
Application of Western Blot?
Confirmation of HIV
Describe Western Blotting (aka Immunoblotting)
- SDS page separates out the proteins on a gel by applying electrical field
- Transfer proteins from gel to nitrocellulose
- Add primary antibody
- Add secondary antibody (*has enzyme tag)
- Gives color
