Denaturation, renaturation of nucleic acids

Question 1

Denaturation

Answer 1

The process of separating the polynucleotide strands of duplex DNA
-melting

Question 2

Renaturation

Answer 2

The joining together by typical base pairing of 2 fully separated complementary sequences, resulting in native duplex structure
-annealing

Question 3

Melting Temperature (Tm)

Answer 3

The temperature at which half of the nucleic acid strands are denatured

Question 4

Hybridization

Answer 4

The process of forming a double helix from 2 complementary ss of nucleic acid
-in lab context

Question 5

Helix–> random coil

Answer 5

• electrostatic repulsion of side chains (charge on phosphate group)
• higher entropy of random coil

Question 6

random coil–>Helix

Answer 6

• H bonding b/w bps

- base “stacking” (van der waals)

Question 7

What factors effect hybrid stability? (4)

Answer 7

1. Base composition-Tm depends on the G+C/A+T ratio, increases w/ increased G+C content
2. Length of nucleic acid sequence
3. Degree of sequence similarity b/w hybrids
4. Temperature-rxn temp. affects the rate of nucleic acid hybridization
   - max rate for DNA-DNA re-association occurs at 25 degree below Tm: the annealing temp.

Question 8

What factors effect hybrid stability?-Degree of sequence similarity b/w hybrids

Answer 8

• The Tm of perfectly matched DNA duplexes in solution depends on various factors (ionic strength, base composition, denaturing agents)
• Every 1% mismatch of bases reduced Tm by 1 degree
• oligos Tm calculated by 4(GC)+2(AT)
• Every mismatch in oligonucleotide reduces Tm by 5 degrees

Question 9

Hybridization Analysis-DNA labeling

Answer 9

• Attachement of radioactive, fluorescent or other type of marker to DNA molecules
  e. g random primer labeling-uses complex mixture of random oligos (hexamers) as primers to initiate integration of radioactive precursors into DNA

Question 10

Random primer labelling

Answer 10

1. denature dna to ss
2. anneal hexanucleotide primer (collection of random hexamers, relying on fact that by chance the primer will bind DNA template
3. extend primers with klenow fragment of DNA pol I in presence of radioactive precursors

Question 11

Hybridization- Detection of labelled molecules

Answer 11

Radioactive
-labeled molecules detected w/ x ray sensitive film (autoradiography) or radiation-sensitive phosphorescent screen (phosphorimaging)
Non-radioactive
-fluorescence: molecules labeled w/ fluorophores (dyes) w/ different emission wavelengths, detected w/ film or fluorescence detector
-chemiluminescence-makes use of rxn b/w label and additional chemicals, rxn generates light, signal detected w/ film

Question 12

Hybridization Analysis-southern blotting

Answer 12

• run nucleic acid on gel
• take separated DNA out of gel, onto solid/semisolid material
• wicking material, buffer solution, weight on top to keep squashed down, paper towel suck liquid from the gel–> capillary action
• end up w/ nitrocellulose replica of gel
• expose UV to cross link the DNA
• incubate w/ probe designed to hybridize to sequence being studied
• now membrane has DNA bound w/ probe
• develop film (autoradiograph)

Question 13

Hybridization Analysis-Northern Blot

Answer 13

• Extract RNA from cells
• run on gel
• carry out transfer and autoradiography
• use DNA probe b/c more stable

Question 14

Steps of Microarrays

Answer 14

1. Isolate mRNAs from cells at 2 stages of development-all the genes expressed in the cell at that stage
2. convert mRNAs to cDNA by RT using fluorescent dNTPs
3. Add cDNA to microarray, anneals to DNA spots w/complementary sequences
4. each fluorescent spot represents a gene expressed in the cell

Question 15

Use of Microarray

Answer 15

• useful for assessing differential genes

- genes that are turned off at 1 point, turned on at another