Week 6 Scott - Molecular Bio Techniques

Question 1

What are the four DNA techniques discussed in class?

Answer 1

1. Restriction enzyme digestion
2. Southern blotting
3. Conventional sequencing
4. PCR

Question 2

What are the three mRNA techniques discussed in class?

Answer 2

1. Northern blotting
2. RT-PCR
3. Microarray analysis

Question 3

What are the two Protein techniques discussed in class?

Answer 3

1. ELISA
2. Western blotting

Question 4

What are the key points on restriction enzyme digestion?

Answer 4

• bind DNA at specific sequences and cleave it
• used to identify DNA
• used to recombine DNA (create “sticky ends”)

Question 5

What is southern blotting used for?

Answer 5

Identify a specific region of DNA.

Target DNA is identified based on restriction fragment size and hybridization to a probe

Question 6

What are the steps in Southern blotting?

Answer 6

1. Denature double-stranded DNA
2. Separate DNA fragments by gel electophoresis
3. Add probe/primer, wash
4. Labeled probe/primer binds to target DNA

Question 7

What is Sanger/conventional DNA sequencing used for?

Answer 7

Gives exact order of base pairs in a piece of DNA.

Identify single base mutations and polymorphisms.

(in vitro DNA replication)

Question 8

What are the steps in Sanger/conventional DNA sequencing?

Answer 8

1. Primer determines start site
2. Fragments are determined by dideoxynucleotides (ddNTPs = chain terminators)
3. Length of terminated fragment tells position of base
4. Fragments separated by gel electrophoresis or labeled with radioactive/fluorescent tags and detected by film/laser

Question 9

What are the key points of PCR?

Answer 9

• Amplifies a defined region of DNA (exponentially)
• combines specificity and amplification
  
  • region of DNA determined by hybridization of short primers

Question 10

What are the clinical applications of DNA techniques?

Answer 10

• Identification of mutations
  
  • familial breast cancer
• Diagnosis of infectious agents
  
  • HPV

(Emerging application = prenatal screening)

Question 11

What is Northern Blotting used for?

Answer 11

• Identify one target RNA in a complex mixture

Question 12

What are the steps of Northern blotting?

Answer 12

1. separate RNAs in cell by size on gel
2. transfer fragments to membrane
3. probe binds to short RNA target
4. labeled probe marks target RNA

*Only going to find one mRNA target, so this test is limited and difficult to quantitatively measure.

Question 13

What is RT-PCR used for?

Answer 13

Adapts PCR to quantitatively measure mRNA levels.

mRNA → cDNA by reverse transcriptase

Add fluorescent dye.

Amplify.

Question 14

What is Microarray Analysis used for?

Answer 14

• 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).

Question 15

What is the microarray analysis technique based on?

Answer 15

• 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.

Question 16

What are the clinical aplications of RNA techniques?

Answer 16

• 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

Question 17

What is the ELISA test used for?

Answer 17

• Detect specific protein in complex mixture
• high output and quantitative

Question 18

What is ELISA based on?

Answer 18

• Specific interaction of protein with antibody
  
  • (no separation by size)
  • HIV testing

Question 19

What are the steps in ELISA?

Answer 19

1. collect patient’s serum (with antibodies)
2. add primary antigen
3. antibodies bind to antigen
4. add secondary antibody with color tag
5. binds to primary antibody (if present)
6. substrate changes color

Question 20

What is Western Blotting used for?

Answer 20

Identify a specific protein in a complex mix

**Target protein is identified by size and specific interaction between antibody and protein.

Question 21

What are the steps in Western blotting?

Answer 21

1. Isolate cellular proteins
2. coat proteins with SDS
3. run proteins in gel electrophoresis (separate by size)
4. transfer to membrane
5. Incubate with primary antibody
6. 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.

Question 22

What are the clinical applications of protein techniques?

Answer 22

• Diagnosis of infectious disease
  
  • Hepatitis C
• Emerging application
  
  • design therapeutic antibodies

Question 23

What are the limitations of next generation sequencing?

Answer 23

Cost still high

Interpretation (sequence differences)

Effective treatments are often not available.

Question 24

What is the clinical potential for RNA interference (RNAi)?

Answer 24

To specifically deplete mRNAs involved in disease.

-most common effect is to inactivate the target mRNA using miRNAs or siRNAs (cleavage, degradation, block translation)