Use of Molecular Genetics in Med. I

Question 1

describe class II restriction endonucleases

Answer 1

• Cut DNA in a precise and reproducible fashion
• Cuts dsDNA within a symmetrical recognition site (normally)
• Hydrolyze phosphate backbone to give a 5’-phosphate and 3’-OH
• Blunt or sticky overhangs a few bp in length
• Enzymes are homodimers that recognize short, symmetric DNA sequences

Question 2

name the 4 DNA pol

Answer 2

1. DNA Pol I (from E. coli)
   
   1. 5’ to 3’
2. Klenow Fragment DNA pol
   
   1. 5’ to 3’ pol
3. Reverse transcriptase (RNA dependent DNA pol)
   
   1. 5’ to 3’ pol and 3’ to 5’ exonuclease
   2. Needs RNA as template
4. Taq polymerase
   
   1. 5’ to 3’ polymerase activity only
   2. Used in PCR (requires primers)
   3. New versions contain proofreding capabilities)

Question 3

describe DNA ligase

Answer 3

• DNA joining enzyme
• Catalyzes the formation of phosphodiester bond
• 5’ phosphate of one nucleotide to the 3’ hydroxyl of the next nucleotide
• Requires ATP

Question 4

name the 3 important features of cloning vectors

Answer 4

1. must be able to self-replicate in a host cell
2. must have a region called a multiple cloning site (MCS)
   
   1. a number of unique restriction sites all in the same region which are unique and not present anywhere else in the vector
3. Must contain a selectable marker
   
   1. typically a gene which confers antibiotic resistance to the host cell
   2. sometimes it is a gene for an enzyme that is not found in host cell

Question 5

describe “cloning” a piece of DNA

Answer 5

• Making multiple identical copies of a fragment of DNA
• Introducing a foreign DNA into replicating cells
• Most commonly, to clone a specific nucleotide sequence of interest
• Reaction involves cutting a fragment of DNA and paste it into a plasmid vector then inserting it into host cell

Question 6

name the principles of PCR

Answer 6

• Heat to denature DNA into single strands
• Cool and allow Primers to anneal to the sequence of interest (primers have free 3’-OH which is required to initiate DNA synthesis)
• Bring to optimum temp for Taq polymerase to allow DNA synthesis in the 5’ to 3’ direction
• Repeat many times

Question 7

name the 6 components for PCR

Answer 7

• Thermal stable polymerase: an enzyme which synthesizes new strand of DNA complementary to an existing single strand of DNA or RNA template in the 5’ to 3’ direction
• Primers: short synthetic oligonucleotides, typically 20 bp which primes DNA synthesis
• dNTPs
• Magnesium chloride
• Buffer
• DNA material to act as a complementary template for new DNA synthesis

Question 8

name the steps of PCR

Answer 8

• Denaturation
  
  • turn ds DNA into 2 ss DNA
• Annealing
  
  • allows primers to anneal to the right complementary strand
• Primer extension
  
  • polymerization by the DNA pol
• Three steps repeated many times

Question 9

how to amplify ss mRNA?

Answer 9

• Making coding DNA (cDNA) from mRNA
  
  • Isolate mRNA by taking advantage of the poly A tail
  • add oligo-dT primer
  • add dNTPs and reverse transcriptase
  • Destroy mRNA
  • generate 2nd strand DNA
  • now use dsDNA for template
  • PCR

Question 10

describe Souther blots

Answer 10

• Probe is created, which is ssDNA that is complementary to the gene region you are looking for
  
  • probe is tagged with either radioactive isotope or fluorescent marker
    
    • presence vs absence of DNA region
    • presence vs absence of point mutation
• DNA is cut with restriction enzymes and separated by GE
• gel is sokaed in an alkaline solution to denature the DNA
• DNA is transferred from gel to DNA-binding membrane
• Radioactive probe is hybridized to the membrane
• Excess probe washed
• Membrane exposed to film
• DNA fragments of interest detected

Question 11

describe Northern blot

Answer 11

• Northern blot is similar to Southern blot, only that the sample contains isolated mRNA molecules that are separated by GE
• Transferred to a membrane
• Hybridized with a radiolabeled probe
• Excess probe is washed off, membrane is dried and exposed to film
• Detects amount and size of the particular mRNA of interest

Question 12

describe Western blots

Answer 12

• Western blot analysis can be used to detect and quantify the amount of a particular protein
  
  • electrophoresis of protein
  • transfer nylon membrane
  • detection using a labeled antibody system (typically an enzyme label and a chemiluminescent substrate)

Question 13

describe Sanger sequencing

Answer 13

• 4 DNA synthesis reactions set up, each with 1 ddNTP and all with template DNA, radio-labeled primer, DNA pol, mixture dNTPs
• DNA synthesis will occur, base pairs incorporated into growing chain until randomly a ddNTP is incorporated
  
  • synthesis will stop since no free 3’OH
• Millions of reactions proceeds, each reactions stops when ddNTP incorporated
• Each reaction is separated by polyacrylamide GE and the gel is later exposed to X-ray film
• Label is on the complementary strand

Question 14

describe RFLP analysis

Answer 14

• Restriction Fragment Length Polymorphism
• Single base pair change in the DNA may cause creation or destruction of a restriction site
• Used to diagnose Sickle-cell or Maple Syrup Urine disease

Question 15

describe ASO (allele specific oligonucleotide) probes

Answer 15

• probes are usually 15-21 bases long and very sensitive
• Complementary to the DNA of interest
• Used to detect polymorphisms or common genetic mutations
  
  • CFTR
  • Sickle Cell
  • Hemochromatosis