Molecular Diagnositics Flashcards
What are two techniques to detect infectious agents and diagnose inherited disorders
- Hybridization
- Polymerase chain reaction
- detection is based on a known pathogen sequence
- diagnosis is based on known Muna genome
Explain how hybridization works
*useful for detection and quantification of target DNA or RNA
- meant to create single stranded oligonucleotides called probes
- ss-DNA binds to another strand of DNA or RNA to form DNA-DNA or DNA-RNA hybrid
TARGET DNA is made to ss-DNA then immobilized on a support and is called blotting
Southern, northern, and western blotting
Southern Blotting vs Northern blotting
Southern blotting- both probe and target nucleic acid are DNA
Northern blotting- probe is as-DNA and target is mRNA
What part of northern or southern blotting is labeled with radioactive fluorescent tag
The ss-DNA probe
Describe the step of blotting technique
- Material is separated by gel electrophoresis (smaller moves faster)
- Transfer to membrane so it can be exposed on the blot
- Add tagged probe to reveal bands of interest (hybridization occurs)
- Solid lines represent bands reactive with probe
- Show on visual: only bands reactive with probe are made visible
What is the purpose of a southern blot
To determine which restriction fragments are associated with a gene
What is the purpose of northern blot
To measure size and quantities of mRNA molecules to answer questions about gene expression
What is the purpose of western blot
*targets protein
To measure the amount of protein or antibody present
Describe the purpose and steps of PCR
Purpose- amplify DNA sample to create multiple copies
- Ds-DNA from pathogen is denatured at high temps to create ss-DMA
- Primers added and anneal to DNA
- Add all 4 dNTPS
- Taq polymerase -synthesizes DNA
What are the advantages and disadvantages of PCR
Advantage- very small amount of template DNA is needed
Disadvantage- you MUST know the sequence of the DNA for primer design
PCR is also called ____
Cell-free cloning
*allows early detection of microorganisms and detection of specific gene mutations
What is qPCR? What is it used for?
Quantitative PCR (or real time PCR)
Used to quantify copy number of a specific gene in two or more samples in real time
[detect levels of infectious agent and determine levels of gene expression)
What is added to qPCR that is not in regular PCR
In addition to primers, qPCR includes probes which flouresce in presence of the PCR product
- probes are usually oligo with a fluorescent tag.
- can measure intensity of light to quantify
What are the molecular techniques used to detect variations in DNA sequence for forensics and diagnostics
- RFLP
2. VNTR
How does RFLP work?
-based off the idea that genes differ in the recognitiion sequence for restriction enzymes
- Sample DNA is added to restriction endonucleases
- DNA is put on gel and southern blot is used with DNA probe
- X-ray film shows bands and if bands match then they are related
How to detect mutation of sickle cell disease using RFLP
Because normal globulin has 3 Ddel restriction sites and those with sickle cell only have 2, then you can add Ddel endonuclease to cleave the DNA and use southern blotting to determine mutation
How does VNTR work and what is it useful for ?
- useful for identification and severity of inherited diseases
- based on short tandem repeats that vary in individuals being isolated from genomic sample by flanking restriction sites or through PCR
- important in diagnosing Huntington’s disease
RFLP and VNTR can be used for diagnostic of disease causing mutations in what three ways?
- Prenatal diagnosis (amniocentesis)
- New born screening
- Carriers of disease
how are recombinant proteins produced
- cDNA of the protein is inserted into an expression vector (plasmid DNA of bacteria cut with restriction enzymes to make plastmid vector)
- Makes recombinant DNA
- Recombinant DNA is added to bacteria and recombinant bacteria multiply then producing the protein
What are important recombinant proteins made
Insulin Growth hormone Erythropoietin Clotting factors Flu vaccine
Normal human insulin has ____ at position 28 and ____ at position 29 at C terminus of B chain
Proline @ 28
Lysine @ 29
What reverse the position of proline and lysine in insulin to make insulin fast acting
Lispro
What insulin drug replaces the 28 position proline to aspartic acid
Insulin Aspart (Novo Nordisk)
T/F you can treat AD with monoclonal antibodies
True
What is an immunlogical technique which tests for the levels of specific antigen or antibody concentrations in biological samples using corresponding antibody or antigen
ELISA
Enzyme linked immunosorbent assay
How does indirect ELISA work ?
*measures amount of antibody
- Has antigens present in solution
- Specific antibodies attach to antigen
- Enzyme-linked antibody binds to specific antibody
- Substrate is added and converted to colored product
- Rate of color formation is proportional to amount of antibody
* washed in between each step
How does sandwich Elisa work
*measures amount of antigen in sample
- Monoclonal antibody present
- Added Antigen binds to antibody
- Second antibody linked to enzyme binds to antigen
- Substrate is added and converted product is colored. Rate of color formation is proportional to amount of antigen
ANTIBODY LOOKS LIKE A Y
What helps diagnosis HIV
Indirect Elisa
- measure HIV antibodies
- confirmation by western blot
What ELISA technique is used for MI detection
Sandwich ELISA
- measures amount of troponin protein (T and I) in an acute MI
- remember antigens are proteins
What ELISA technique is used in pregnancy tests
Sandwich ELISA.
-Free hCG antibodies bind to hCG antigens in urine
How does western blotting (immunoblotting) work to see if viral proteins are present ?
- SDS-PAGE serperates proteins on a gel
- Transfer proteins to nitrocellulose to expose to surface
- Add primary antibody
- Add secondary antibody with enzyme tag
- Add substrate which gives color
What confirms HIV diagnosis
Western Blot
When would restriction occur in G1?
If growth factors are limiting
*progression past RP is growth factor -independent
What does the G1 checkpoint respond to
DNA damage
What does the G2 checkpoint respond to
Verify complete genomic duplication
What does the metaphase checkpoint check for
Ensures chromosomes are attached to mitosis spindle
