Lecture 4 + 5: Denaturation/ Renaturation of nucleic acids

What is Tm affected by? (7)

-base composition
-sequence
-length
-salt concentration
-organic solvent concentration
-pH
-NA source: RNA/RNA > RNA/DNA > DNA/DNA

What does the hyperchromicity curve show?

shows the absorbance @ 260 nm increases as dsDNA turns into ssDNA

How can we monitor DNA denaturation?

by spectrophotometric measurement of absorbance at 260 nm

Tm = ? - ?

Tm = melting point of a NA - mid-point of the hyperchromicity curve

The hyperchromicity curve is a linear relationship between what two things?

linear relationship between GC content and Tm

Why is the DNA melting curve so steep?

because as the temp increases the bases start to unpair/ repair and as soon as the Tm is reached the bases unpair all at once

Which regions in a DNA strand melt first?

AT rich regions

What are the significance of AT rich regions? (what are they usually sights for)

usually sights for transcription/ replication are AT rich

Is melting dependent on [DNA]?

no, DNA that is separating or not does not influence other DNA from separating or not

Why does complete denaturation of genomic DNA require incubation at 94 degrees celsius

ensures complete separation because all DNA separates below 94 degrees

How does the NA source affect Tm? (what has a higher Tm: RNA, DNA, or hybrid duplexes)

RNA/RNA > RNA/DNA > DNA/DNA

How does DNA sequence affect Tm?

repeats of either GCGC or ATAT have a lower Tm than random bases
- ~20 degrees different

How does DNA length affect Tm?

-DNA length effect is primarily on dsDNA

All oligonucleotide primers have a unique Tm that is what?

that is <

dsDNAs longer than ~100 bp must be heated to what to ensure complete denaturation?

94 degrees celsius

How does salt concentration affect Tm?

as [ion] increases, so does Tm
-salt stabilizes the DNA duplex by shielding the repulsion of PO4- in backbones

A 10-fold increase in [Na+] increases Tm by how many degrees celsius?

16.6 degrees celsius

What salt (cation) has the strongest stabilizing effect?

Mg2+

Why wont DNA denature in water?

because of low ionic strength

Why is DNA less stable in water at 4 degrees than if it were in water at -20 degrees?

because of acidic H2O and/ or contaminating nucleases

Why is DNA more stable in TE (EDTA)?

-EDTA binds to divalent cations (Mg2+)
-all DNA binding proteins need Mg2+ for their function
-so if there isn't any Mg2+ for DNA binding proteins to use for their function, proteins like exonucleases cannot cleave DNA, leaving DNA more stable

How do organic solvents effect Tm?

-decreases Tm
-contain functional groups that can form H-bonds with the bases and mimic base pairing
-ssDNA can be base paired to a solvent and then the DNA molecule is more stable

How would you remove organic solvents that are base paired to a DNA strand?

by providing a strand that has 100% complementarity - it will favour base pairing over the organic solvents

How does pH affect Tm?

-decreases Tm
->11.3 = DNA cannot be dsDNA. excess OH- removes the H+ forming the base pairing thus ionizing the H-bonds and interferes with base stacking thereby separating the strands

What is the goal of all NA renaturation?

to only form a double stranded molecule with all perfect Watson-Crick base pairing

What are the two steps to NA renaturation called?

1) nucleation
2) zippering

What is nucleation?

random short complementary regions form where two short complementary sequences bind

What is zippering?

if the adjacent bases are also complementary rapid base pairing occurs

How fast is rapid zippering?

>1000 bp/s

What is the rate limiting step in NA renaturation?

nucleations

How does Tm (desired conditions) effect the rate of NA renaturation?

decreases the rate

How does Tm (desired conditions) effect the specificity in NA renaturation?

increases the specificity

How does temp above Tm effect the rate of NA renaturation?

no annealing

How does temp above Tm effect the specificity in NA renaturation?

zero specificity

How does temp way below Tm effect the rate of NA renaturation?

increases the rate

How does temp way below Tm effect the specificity in NA renaturation?

decreases the specificity

How does high [organic solvents] effect the rate of NA renaturation?

decreases the rate

How does high [organic solvents] effect the specificity in NA renaturation?

increases the specificity

How does low [salt] effect the rate of NA renaturation?

decreases the rate

How does high [salt] effect the specificity in NA renaturation?

increases the specificity

Why does high [salt] decrease the rate and increase the specificity in NA renaturation?

-high salt stabilizes the DNA strands be reducing the repulsion between the two strands
-less repulsion means more base pair interactions which lowers the specificity

How does high [DNA] effect the rate of NA renaturation?

increases the rate

How does high [DNA] effect the specificity in NA renaturation?

decreases the specificity

What two things increase stringency?

-raising temp: Tm+
-lowering [salt] during hybridization or washes

What are the two methods we can use to calculate Tm?

-spectrophotometrically
-mathematically

What are the names of the 3 formulas used to calculate Tm?

-Marmur formula
-Wallace formula
-Duplex formulas

What is the Marmur formula?

Tm = 4(GC) + 2(AT)

What does the Marmur formula take into account? (1)

only takes into account the effect of GC content on Tm

What is the Marmur formula valid for? (in what situation would we use the Marmur formula?) (2)

-oligos

What does the Wallace formula take into account? (2)

the GC content and length

Why are the duplex formulas more complex than the Marmur and Wallace formulas?

because they take into account more parameters to what keeps the duplex together

What are the 4 parameters taken into account when calculating the Tm using the more complex duplex formulas?

-[salt]
-GC content
-length
-[organic solvents]

What are the two criteria required in order to use the simplified DNA-DNA duplex formula?

-for lonf DNA duplexes in 0.3M Na+
-in the absence of formamide

What other formula does the simplified DNA-DNA duplex formula look similar to?

the Wallace equation

Why is one of the criteria to use the simplified DNA-DNA duplex formula to have long duplexes in 0.3M Na+?

because 0.3M Na+ is the normal/ standard physiological buffer that genomic DNA exists in

Why do the formulas differ for the different NA duplexes?

because the duplexes have different strengths in terms of staying together as a duplex: RNA:RNA > RNA:DNA > DNA:DNA