GENET 390: Topic 2.3 - Denaturing + Renaturing DNA + Calculating Tm

What bonds are being broken during denaturing of DNA?

H-Bonds between base pairs

What is the easiest way to denature DNA

Heating it

***At what temp is all DNA fully denatured?

90 degrees Celsius

What occurs during the process of renaturation/annealing?

Reformation of H-Bonds

How do you reanneal DNA?

REMOVE the thing that is causing the breaking of bonds

• eg. Lower temp

• eg. Neutralize base (seen with mini prep)

Define annealing temp

Temp at which a particular sequence reanneals

How does High Annealing temp affect stringency? define stringency

High Stringency = High specificity in base pairing

• Higher annealing temp = Higher base pair specificity

  • reduces non-specific binding

Why does Higher temps lead to increased stringency?

Mismatches will fall apart at lower temperatures

What is a Hyperchromicity curve? How does it relate to denaturation/renaturation

Measures Absorbance at 260nm

• Measure DNA denaturation by spectrophotometric measurements

****How does ABSORBANCE change as DNA denatures?

Absorbance INCREASES as dsDNA —> ssDNA

• ssDNA absorbs more

What is Tm?

Melting temp. Temp at which DNA denatures

At what point along the hyperchromicity curve is Tm measured at?

MIDPOINT

TRUE or FALSE: Base composition does not affect denaturation

FALSE

• G-C bp = more stable at higher temps

Why are GC pairs more stable?

BASE stacking (driving force)

• also a little bit to do with H-bond #

How does Tm change as GC content increases?

Tm Increases

***Although DNA fully denatures at 90 degrees Celsius, what temp do we practically use to ensure all dsDNA is denatured?

94 degrees Celsius

• to accommodate for GC content effects

***What is the restriction on DNA size (bp) for 94 degrees denaturation?

• not too sure on this 100bp thing

100 bp = minimum

***Why not use temps higher than 94?

Any higher = Increase risk of breaking Phosphodiester bonds

• heat breaks all bonds

• Weaker bonds are just easier to break at lower temps

*****What are the 6 FACTORS that influence Tm besides the GC content?

1. Sequence length

2. Presence of Salts

3. Presence of Organic solvents

4. pH

5. Nucleic acid type (DNA vs. RNA)

6. Sequence homology

1. How does sequence length affect Tm?

• Does Tm become more dependent on composition in SHORTER or LONGER sequences?

More dependent on composition in Shorter Sequences = Much more variable in Tm

• VERY dependent on GC content

Longer sequences = less affected by CG content

Around how many bp is Tm only slightly affected by composition?

greater than 1000

2. How does Salt affect Tm

Presence of salt INCREASES Tm

How does salt increase Tm

Ionic strength (presence of salt) stabilizes DNA duplex as part of the HYDRATION SHELL

• Shields the repulsion of phosphates in the backbone

How does Ion concentration affect Tm?

Increase [ion] = increase stability = Increase Tm

***Are DIVALENT or MONOVALENT cations preferred for salt stabilizing effects on DNA?

DIVALENT

• eg. Mg 2+

3. How do Organic Solvents Effect Tm?

Decreases Tm

**How/why do Organic solvents Decrease Tm?

Some can act as COMPETITIVE DENATURANTS

• Molecules contain functional groups that can compete with base stacking interactions = Destabilize double helix

• Disrupts the H-Bonds

Not all organic solvents disrupt Base stacking: what are some that do? (4)

1. Urea

2. Formaldehyde

3. Formamide

4. DMSO

4. How does pH affect Tm?

Both low + high pH = Decreases Tm

• saw in miniprep with basic solutions

How do Basic solution decrease the Tm?

OH from base denatures DNA by removing the H+ of the H-bond forming the Base Pairing = Break bond

• IONIZE NITROGENOUS BASES

BASE = STRONG H-ACCEPTOR

• pH greater than 9

5. How does Type of nucleic acid affect Tm?

Higher Tm

1. RNA:RNA

2. RNA:DNA

3. DNA:DNA

Lower Tm

Why is Tm RNA:RNA > RNA:DNA > DNA:DNA? (2 reasons)

1. RNA = Capable of forming 2x’s the H-BONDS between H2O + Backbone

   • due to 2’ OH = More stable double helix when in H2O

2. 2’OH = Stiffens the Helix = Strengthen double Helix

6. How does Sequence Homology affect Tm? DEFINE SEQUENCE HOMOLOGY

Definition: Amount of similarity between sequences/ amount of correctly matched based pairs

Low Homology (any MISMATCH) = SUBTANTIAL DECREASE in Tm

eg. 1 mismatch in DNA:DANA duplex = drop Tm by 20 degrees

Are there times where we will tolerate mismatches?

Yes

• Finding relative sequences in southern

Why is the DNA melting curve so steep?

Once you get it started its a lot easier to pull apart

Why is it a lot easier to pull apart DNA after its started?

The initial pulling apart Weakens neighboring Base Pairs

Which regions melt first?

A/T rich

***Is Melting dependent on DNA CONCENTRATION? Why or why not?

YES!

• High Concentrations = favor DUPLEX FORMATION = Increases Tm

  • Duplex formation is ssDNA —> dsDNA

    • higher concentration = more likely to find match to reanneal???

Why does Complete Denaturation of Genomic DNA require incubation at 94 degrees Celsius?

94 will denature a sequence that is 100% G/C pairs

• Safe temp to ensure complete denaturation WITHOUT strand breakage

What occurs if you set up annealing conditions incorrectly?

Mismatches in annealed duplex

How many STEPS are required for Renaturing?

2

What are the 2 STEPS of renaturation

1. Nucleation

2. Zippering

Which of these 2 steps = the rate limiting step? WHY?

Nucleation

• Its the hardest to START/ takes a long time

  • once you get it started the process = quick

****What is Nucleation?

RANDOM short complementary sequence formations

***How do these short complementary bp sequences form?

BROWNIAN MOTION

• Random collision between sequences

***What is the required minimum length of bp needed to start zippering?

3 base pairs

Define Zippering

Rapid base pairing

When does Zippering occur?

if Bases ADJACENT TO the 3 bp are ALSO COMPLEMENTARY

What occurs if the Adjacent base pairs are not complementary?

Zippering does not occur + base pairing dissociates

**True or False: Only one nucleation event occurs and then zippering occurs immediately in succession

FALSE

• MANY are require

  • TRIAL + ERROR over and over until the 3 bp are matched to allow zippering + zippering = possible

Is/Should Zippering be VERY sequence specific?

Yes

• However specificity Depends on conditions

What are the conditions for Renaturation?

OPPOSITE of DENATURATION

What are the opposite conditions (4)

1. Low temp (below Tm)

2. HIGH formaldehyde/formamide

3. Some salt

4. High DNA concentrations

****What are the optimal Reannealing Temp?

20-25 degrees below Tm

• Lower temps = allow Toleration of Mismatch

****What temps Ensure STRINGENCY/SPECIFICITY?

Close to Tm

• Higher temps

How does salt help with reannealing?

Prevent electrostatic repulsion between backbones

• increase stability of duplex

How does High [DNA] help with reannealing?

2 molecules are required for rate limiting nucleation step = reannealing is concentration dependent

• higher [ ] more likely for random motion to result in correct bp

Can DNA [ ] ever be too high? Why or why not?

Yes

• Too high = form Concatemers

Define Concatemers

Multiple sequences all annealed

***High stringency is always desired: What 3 conditions promote high stringency?

1. Higher temps closer to Tm (Still has to be below)

2. Keep salt concentration low

3. High formaldehyde/formamide

Compare the speed of reannealing to denaturation?

Slow compared to denaturation

• several hours under lab conditions

***What type of DNA reanneals faster? (what characteristics)?

SHORTER or HIGHLY REPETITIVE reanneals faster than Long, non-repetitive DNA

Finish the Sentence: Using the rate of reannealing you can measure the frequency of _____

specific base sequences

What are 4 examples of where we use reannealing (not sure how important)

• Southern + Northern (use probes)

• PCR (Primers)

• Cloning (variety)

****What is one example for when it is important to CALCULATE Tm?

Tm = important consideration for Primer design

• normally calculated by software though

What are the 2 main ways to calculate Tm?

1. Basic

2. Nearest Neighbors

What are the 2 equations used in BASIC calculations? What type of sequences are they each applied to? (not sure how important)

Marmur: SHORT less than 14 bp =

• Tm = 4xGC + 2xAT -7

Wallace: LONGER more than 14 bp

The above applies to DNA:DNA + DNA:RNA duplexes. WHAT ABOUT RNA:RNA duplexes Are the same calculations applicable? Why or Why not?

NO - RNA:RNA = stronger association = Stronger duplex

Calculations of Tm = DIFFERENT depending on NUCLEIC ACID TYPE