Molecular Genetic Diagnosis

Question 1

Restriction enzymes

Answer 1

• cut at SPECIFIC sequence (palindromic sequences- symmetry)
• Either staggered or blunt cut
• Used in molecular cloning and blot tests

Question 2

Steps of Molecular Cloning

Answer 2

• 1- digest vector DNA AND and the target DNA you want to be cloned into small pieces (w/ same restriction enzyme)
• 2- join pieces w/ DNA ligase
• 3- introduce into host cells (ex- E coli) –> growth

Question 3

Steps of Southern Blot

Answer 3

• 1- Target DNA is digested via restriction enzyme –> large # fragments
• 2- Fragments sep by gel electrophoresis (by size or charge)
• 3- Denature DNA and transfer fragments to membrane
• 4- Hybridize w/ PROBE (probe is composed of specific DNA sequences)
  
  • The probe is in solution
  • Probe must have similar sequence to target DNA min your sample but does not have to be identical

Question 4

Uses of Southern Blot Test

Answer 4

• Determine presence or absence of gene

- Determine if genomic loci is modified (insertion/deletion)

Question 5

Northern Blot

Answer 5

• Use nucleic acid probe
• examines RNA not DNA
• So determined if gene is EXPRESSED in particular tissue

Question 6

Western Blot

Answer 6

• Use antibody probe (antibody against target protein will bind that protein)
• examines proteins
• used to determine amount, presence/absence and molecular weight of a protein

Question 7

When is ASO Hybridization used?

Answer 7

• Used when location of target sequence is known (ex- known mutation of sickle cell or CF)
• SENSITIVE - can pick up a single base pair mismatch

**as opposed to other blots

Question 8

PCR Steps

Answer 8

• 1- Heat/denature DNA to sep 2 strands
• 2- Cool DNA which allows primers to surround target DNA
• 3- Heat-stable DNA polymerase –> DNA synthesis (produce mult copies-amp)
• 4- Analyze PCR DNA product using gel electrophoresis

Question 9

PCR Uses

Answer 9

• Amplify small pieces of DNA
• Detect small changes in DNA (few BPs)
• Diagnosis, forensics, etc

Question 10

Limitations to PCR

Answer 10

• Can only be used for sequences up to a certain size b/c the special DNA polymerase used will eventually fall off
• Must know the DNA sequence that you want to amplify
  
  • You create the primers yourself

Question 11

RT-PCR

Answer 11

used to amplify DNA from an RNA sample (as opposed to from a DNA sample like normal PCR)

**reverse transcriptase followed by DNA polymerase

Question 12

Steps of Microarray Analysis

Answer 12

• 1- start w/ 2 mRNA samples and convert each to cDNA
• 2- dye ea sample a different color
• 3- hybridize both samples to same microarray
• 4- wash and then scan to see color of ea dot

Question 13

Uses of Microarray

Answer 13

• Monitor the expression of thousands of genes simultaneously
• Study differential expression of genes b/n 2 samples
• ID variations in copy number of specific genomic sequences
• ID additions or deletions of sequences in chromosome

Question 14

Proteomics

Answer 14

• Goal= compare proteins of 2 samples
  
  • Protein expression, structure and interactions
• Label ea protein sample w/ dye and compare using gel elcetrophoresis and mass spectrometry

Question 15

CRISPR

Answer 15

• Not currently used in humans but POTENTIAL
• Uses
  
  • Gene targeting and regulating gene expression
    
    • Possibilities (use a guide RNA made by experimenter to…)
      
      • Inactivate gene by double strand break (nonhomologous end joining –> deletion of unwanted gene)
      • Introduce new activator to turn gene ON
      • Introduce new repressor to turn gene OFF
  • Can also learn about gene functions by inactivating genes and see what is lost (“study gene function”)