Week 6 Scott - Molecular Bio Techniques Flashcards
What are the four DNA techniques discussed in class?
- Restriction enzyme digestion
- Southern blotting
- Conventional sequencing
- PCR
What are the three mRNA techniques discussed in class?
- Northern blotting
- RT-PCR
- Microarray analysis
What are the two Protein techniques discussed in class?
- ELISA
- Western blotting
What are the key points on restriction enzyme digestion?
- bind DNA at specific sequences and cleave it
- used to identify DNA
- used to recombine DNA (create “sticky ends”)
What is southern blotting used for?
Identify a specific region of DNA.
Target DNA is identified based on restriction fragment size and hybridization to a probe
What are the steps in Southern blotting?
- Denature double-stranded DNA
- Separate DNA fragments by gel electophoresis
- Add probe/primer, wash
- Labeled probe/primer binds to target DNA
What is Sanger/conventional DNA sequencing used for?
Gives exact order of base pairs in a piece of DNA.
Identify single base mutations and polymorphisms.
(in vitro DNA replication)
What are the steps in Sanger/conventional DNA sequencing?
- Primer determines start site
- Fragments are determined by dideoxynucleotides (ddNTPs = chain terminators)
- Length of terminated fragment tells position of base
- Fragments separated by gel electrophoresis or labeled with radioactive/fluorescent tags and detected by film/laser
What are the key points of PCR?
- Amplifies a defined region of DNA (exponentially)
- combines specificity and amplification
- region of DNA determined by hybridization of short primers
What are the clinical applications of DNA techniques?
- Identification of mutations
- familial breast cancer
- Diagnosis of infectious agents
- HPV
(Emerging application = prenatal screening)
What is Northern Blotting used for?
- Identify one target RNA in a complex mixture
What are the steps of Northern blotting?
- separate RNAs in cell by size on gel
- transfer fragments to membrane
- probe binds to short RNA target
- labeled probe marks target RNA
*Only going to find one mRNA target, so this test is limited and difficult to quantitatively measure.
What is RT-PCR used for?
Adapts PCR to quantitatively measure mRNA levels.
mRNA → cDNA by reverse transcriptase
Add fluorescent dye.
Amplify.
What is Microarray Analysis used for?
- Global analysis of mRNA levels.
- Identify individual mRNAs that change in disease states.
- Identify patters of mRNAs expressed in different diseases.
- Do NOT need to know what will change ahead of time (before performing test).
What is the microarray analysis technique based on?
- hybridization of cDNA pop’n derived from sample mRNAs to grid containing known DNAs at specific locations.
- pattern of fluorescence on chip tells which mRNAs are present in each pop’n and relative levels.
What are the clinical aplications of RNA techniques?
- Identification of genetic alterations that affect protein coding regions
- fusion genes in leukemias and lymphomas
- Identification of infectious agents
- screening blood pool for HIV
- determine risk of recurrence in breast cancer
- breast cancer stratification
What is the ELISA test used for?
- Detect specific protein in complex mixture
- high output and quantitative
What is ELISA based on?
- Specific interaction of protein with antibody
- (no separation by size)
- HIV testing
What are the steps in ELISA?
- collect patient’s serum (with antibodies)
- add primary antigen
- antibodies bind to antigen
- add secondary antibody with color tag
- binds to primary antibody (if present)
- substrate changes color
What is Western Blotting used for?
Identify a specific protein in a complex mix
**Target protein is identified by size and specific interaction between antibody and protein.
What are the steps in Western blotting?
- Isolate cellular proteins
- coat proteins with SDS
- run proteins in gel electrophoresis (separate by size)
- transfer to membrane
- Incubate with primary antibody
- incubate with secondary antibody
**Protein is identified based on size and specific interaction with primary antibody.
***Location of protein and primary antibody is detected by secondary antibody.
What are the clinical applications of protein techniques?
- Diagnosis of infectious disease
- Hepatitis C
- Emerging application
- design therapeutic antibodies
What are the limitations of next generation sequencing?
Cost still high
Interpretation (sequence differences)
Effective treatments are often not available.
What is the clinical potential for RNA interference (RNAi)?
To specifically deplete mRNAs involved in disease.
-most common effect is to inactivate the target mRNA using miRNAs or siRNAs (cleavage, degradation, block translation)
