Forces That Destabilize the Double Helix Flashcards
The Two Forces
- heat
- negative charge on phosphate in backbone
- affect kinetics of dissociation and reassociation of DNA molecules - basis of experiments in molecular genetics.
heat
thermal energy increases the mobility of molecules tending to denature (unwind) the two DNA strands.
negative charges on phosphate in backbone
- the repulsive forces of the negatively charged phosphates on the DNA backbone tend to push the two DNA strands apart.
DNA absorbance spectrum
- made in a spectrophotometer by passing light of different wavelengths through a solution of DNA and measuring absorbance
- relative max absorbance at 260 nm
- relative min absorbance at 230 nm
hyperchromicity
- as double stranded DNA denatures
- 40% increase in A260
- further absorbance if single-stranded DNA broken into nucleotides - residual hyperchromicity
From ssDNA to dsDNA
Divide by 1.4
Tm
- thermal denaturation midpoint
- point at which DNA is 1/2 double stranded and 1/2 single stranded
- referred to as melting temp
from Tm to ssDNA
1.4/1.2 Tm
Effect of GC and Cations
- the higher the GC content, the higher the TM. Higher GC content makes a more stable helix and it takes more energy to disrupt the helix.
- the higher the monovalent cation concentration (Na+) the higher the Tm
Why to Effect of GC and Cations
- the Tm of GC rich DNA is higher primarily because the HYDROPHOBIC STACKING INTERACTIONS ARE GREATER in GC rich DNA and secondarily because the HYDROGEN BONDS are stronger in GC pairs than in AT pairs
- Salt stabilizes the double helix because the cations shield (or buffer) the repulsive forces of the negatively charged phosphates - forces that tend to destabilize the helix.
higher the salt
- the more stable the interaction
- more heat to denature
NaCl, GC, Stability, and TM
- if NaCl increases
- and/or GC increases,
- Stability increases
- and TM increases
- more energy to melt double helix.
Nucleic Acid Hybridization
- Effects of GC content and salt concentration have implication for nucleic acid hybridization
hybridization with heterologous DNA or with a heterologous probe
- often DNA-DNA hybrids are made when a strand of DNA from one source hybridizes with DNA strand from another source
- such hybrids often have mismatches - places where sequences are not perfectly complimentary
Dealing with mismatches
- make the mismatched hybrids more stable so that you could detect them.
- prevent hybridization of mismatched hybrids
- make the mismatched hybrids more stable so that you could detect them.
- use gene from one organism to find same gene in DNA of another
- there would be mutations so the genes would not be identical but you could still find them if you did hybridization under conditions that tolerated a few mutation
- prevent hybridization of mismatched hybrids
- make less stable so you would not detect hybrid DNA molecules with mismatches
- find a specific gene among a group of similar genes in an organism to prevent hybridization of DNA except those that match your probe - do this by manipulating.
Manipulate the stringency
- salt concentration
2. temperature
Raising salt concentration
- makes the hybrids more stable which lowers stringency
- allows formation of imperfectly matched hybrids
Lowering salt concentration
- raises stringency
- make hybrids less stable
- mismatches less well tolerated.
lowering the temperature
- makes the hybrids more stable which lowers the stringency
raising the temperature
- raise stringency.
- hybrids with mismatches melt more easily
- are less stable.
When trying to find the gene from a homologous organism
lower the stringency
When trying to find a specific member of a gene family
raise the stringency
blot name
- named for the molecule blotted, not the probe
Southern blot
- the molecule blotted is DNA
- The probe can be DNA, RNA, or anything else that recognizes DNA
- named for inventor Ed Southern
Northern blot
- molecule blotted is RNA
- The probe can be DNA, RNA or anything else that recognizes RNA
Western blot
- proteins are blotted
- the probe can be anything that will recognize a specific protein
- generally antibodies.
when looking for specific mRNA
- blot and use high stringency conditions
- not a tight and well-defined band because of poly-A tails of varying length so bands are broad and smeary
- mRNA from different family members can have different sizes.
Hybridization conditions
- Tm-15 (higher stringency)
- Tm-25 (lower stringency)
- stringency as how many degrees below Tm the hybridization is carried out.
common practice
- add a denaturing element to hybridization mixture
- fomamide common - makes hybrid less stable, thus increases stringency
