Lect 4 Molecular Diagnostics Flashcards
What Techniques are used to Detect Infectious Agents and Diagnose Inherited Disorders?
What is the knowledge assumption for both techniques?
Hybridization & Polymerase Chain Reaction (PCR)
The sequence of the pathogen or the sequence of the gene is known
Hybridization
_ binds to another strand of DNA/RNA with complementary sequence to form _ or _
What is it useful for?
What are probes?
ssDNA binds to DNA/RNA –> DNA-DNA or DNA-RNA Hybrid
Detect/Quantify target DNA/RNA in sample with other DNA/RNA
single stranded oligonucleotides
What are the probe and target nucleic acids in Southern Blotting and what is its purpose?
What are the probe and target nucleic acids in Northern Blotting and what is its purpose?
What is the purpose of Western Blotting?
What are probes labeled with?
- Southern: Probe and target nucleic acid are DNA
- Determine which restriction fragments are associated with a gene
- Northern: Probe is ssDNA and target is mRNA
- Measure size and quantities of mRNA molecules
- Western: Measures amount of protein/antibody
- Radioactive or fluorescent tags
PCR
Why is DNA subjected to high temperature and then cooled?
What is used to synthesize copy of DNA by extending primers?
What is the advantage?
What is the disadvantage?
- dsDNA separated to ssDNA (Denature) –> Primers complementing sequences flank each end of DNA –> Strands anneal as temperature cools
- Taq Polymerase adds dNTPs
- Very small amount of template DNA needed; 109-fold amplification
- Need to know sequence of flanking DNA for primer design; error prone
Quantitative PCR (qPCR)
What is the copy number?
What is it used for?
What does this help determine?
This technique includes something else in addition to primers
What is the significance of cancer cells?
- Copy number - amount of target sequence being amplified
- Quantify copy number of specific gene in two or more samples in real time
- Detect levels of an infectious agent & Determine levels of gene expression
-
Probe (fluoresces only in presence of PCR product)
- Complementary oligo with a fluorescent tag
- Cancer cells have higher amount of target DNA
Restriction Fragment Length Polymorphism (RFLP)
How do individual genomes differ?
What is it used for?
What blotting technique is used?
-
Individual genomes differ by 1 in every 1000 base pairs
- Some occur in recognition sequences for restriction enzymes
- DNA finger printing, Forensic analysis, Paternity testing, Disease detection
- Southern Blotting
RFLP: Detection of Mutations
How many restriction sites on normal B-globulin allele? on patients with sickle cell?
Normal: 3 Ddel restriction sites
Sickle Cell: 2 Ddel restriction sites
Variable Number of Tandem Repeats (VNTR)
What does STR mean?
What are VNTR useful for?
- Short Tandem Repeats (STR) occurs in genome but varies in individuals
- Identification and severity of inherited diseases (Huntington)
How can you tell if someone has Huntington’s based on the genomic sequence?
Increased CAG repeats
Recombinant Proteins
Provides large scale production of what types of proteins?
How are the proteins formed?
- Insulin, Growth Hormone, EPO, Clotting Factors, Vaccines against diseases
- cDNA of the protein inserted into expression vectors
List the Recombinant Protein Steps
- Gene for protein is isolated
- Plasmid DNA extracted from bacterium and cut with restriction enzyme, forming plasmid vector
- Gene inserted into bacterial plasmid vector to form recombinant DNA
- Recombinant DNA introduced into bacterial cell to form recombinant bacterium
- Recombinant bacteria multiply in fermentation tank and produce protein
- Protein extracted, purified, and packaged
Production of antibodies are used for what?
Drugs to treat diseases like cancer, RA, Crohn’s disease, Hepatitis
Enzyme Linked Immunosorbent Assay (ELISA)
What does it test for?
Indirect ELISA and example
Sandwich ELISA and example
- Levels of specific antigen/antibody concentrations in biological samples using corresponding antibody/antigen
- Measures amount of antibody in sample
- Diagnosing HIV
- Measures amount of antigen in sample
- MI Detection and Pregnancy test
Western Blotting aka Immunoblotting
What is used for?
What is the process?
Clinical application?
- Detect levels of target protein in biological sample consisting of mixture of proteins
-
SDS-PAGE used, proteins migrate based on their size by applying electrical field
- Add primary and secondary antibodies (enzyme tag)
- Confirmation of HIV
