Bio200: Cellular Biology
DNA Analysis and Repair
Key Terms:
Taq DNA Polymerase
Gel electrophoresis
Autoradiography
Blot
Blocking
Probe
AP site
Thymine dimer
Proofreading
Base-Excision Repair
Nucleotide-Excision Repair
Mismatch Repair
Key Figures: Figures 6-14, 6-22, 10-3, 10-5, 10-15 and 10-16
Lecture Outline:
Polymerase Chain Reaction (PCR)
replication in a test tube
specific oligonucleotide primers select the region to be copied (or “amplified”)
two primer’s 3’ ends must face each other
no Primase; the artificial primers serve this function
use heat to denature the DNA (thus, no helicase needed)
cycles of heating (to denature) and cooling (to allow primers to bind)
exponential amplification of DNA copy number
because product of one cycle can be the substrate for future cycles
Taq DNA polymerase is isolated from a heat-loving microbe, so it doesn’t denature at high temps
requires an additional mid-level temp for Taq DNA pol to elongate the primers well
Gel Electrophoresis of Nucleic Acids
movement of DNA (or RNA) through a matrix (gel)
driven by an electrical field
small molecules move fastest; large molecules move slowest
detect DNA either with ethidium bromide (binds DNA and fluoresces)
can be easily used to determine size and quantity of DNA
Southern Blot
to identify one specific sequence of DNA among many on a gel
separate DNA (usually total genomic DNA) on a gel
transfer to a membrane (blot)
Block additional binding sites with nonspecific nucleic acid
denature the DNA (ie, make it single stranded)
add a radioactive probe that will basepair to your sequence of interest; allow to renature
wash off nonspecifically bound probe and detect by autoradiography
Types of DNA Damage
Polymerase errors. Misincorporations happen around 10-4
Spontaneous DNA damage
deamination of Cytosine to Uracil
deamination of Adenine to Hypoxanthine (base-pairs like a G)
depurination and loss of base (but sugar-phosphate backbone is intact). Apurinic (AP) site
Induced DNA damage (mutagens!)
Thymine dimers between adjacent T’s on the same strand. caused by UV. makes a cyclobutane
alkylation or other groups covalently added
single- and double-strand breaks caused by radiation
DNA Repair
Proofreading by DNA polymerases
Direct Reversal of DNA Damage
Base-Excision Repair (BER). often used for removing Uracil in DNA
base is cut away from the deoxyribose. AP site (apurinic or apyrimidinic site)
AP Endonuclease cuts the backbone. another enzyme removes the deoxyribose
DNA polymerase + Ligase to fix the gap (different DNA pol than main replication)
Nucleotide Excision Repair (NER). often used to fix to Thymine dimers
cuts backbone on either side of the damage. A helicase removes the damaged strand
DNA polymerase + Ligase to fix
Mismatch Repair (MMR). principally used to fix misincorporations by DNA pol
in E. coli, MutS binds the mismatch site, recruits MutH and MutL proteins
MutH is an endonuclease cutting unmethylated DNA at GATC
MutS/L act as a helicase to remove the strand
gap is repaired by DNA pol and Ligase