Denaturation, renaturation of nucleic acids

denaturation

the process of separating the polynucleotide strands of duplex nucleic acid

sometimes referred to as melting

renaturation

restoration of typical base pairing of two fully-separated complementary sequences, resulting in native duplex structure

sometimes referred to as annealing

melting temperature (Tm)

temperature at which half of the nucleic acid strands are denatured

hybridization

process of forming a double helix from two complimentary single strands of nucleic acid (can be DNA, RNA, or DNA-RNA)

does not require 100% complementarity

What causes a helix to form a random coil?

electrostatic repulsion of side chains (charge on phosphate group)

higher entropy of random coil

What causes a random coil to form a helix?

hydrogen bonding between base pairs

base “stacking”—i.e. can der Waals interactions

What factors affect stability of double-nucleic acid?

base composition, length of sequence, number of mismatches between strands, salt concentration, other factors

How does base composition affect nucleic acid stability?

Tm depends on the G+C/A+T ratio

it increases with increased G+C content

How does length of sequence affect nucleic acid stability?

length of the base-paired region of a nucleic acid duplex affects its thermal stability

How does number of mismatches between strands affect nucleic acid stability?

unpaired bases in double helix lower its stability

How does salt concentration affect nucleic acid stability?

the higher (more cations), the higher the stability

What other factors affect nucleic acid stability?

pH, concentration of nucleic acids themselves, denaturing agents, etc.

general equation for DNA Tm

64.9 + 41x([%G + C]/100 - 16.4/L)

at 50 mM NaCl with 50 nM oligonucleotide concentration at pH=7

%G + C is the percent G+C composition in the nucleic acid, L is the length in nucleotides of the DNA

Tm of very short duplexes (14-20 base pairs)

4 degC (G + C) + 2 degC (A + T)

for those formed by base-paired oligonucleotides, perhaps

for L=20, one mismatch is a 5% mismatch and lowers the Tm by 5 degC

every 1% mismatch of bases in a short DNA duplex redues the Tm by

about 1 degC

reaction temperature and nucleic acid stability

affects the rate of nucleic acid hybridization

annealing temperature

maximum rate for DNA-RNA re-association occurs at ~25 degC below the Tm

hybridization analysis

gene detection and mapping, gene expression studies, etc.

includes Southern blotting, Northern blotting, microarrays

complex nucleic acid sample are fixed to solid surfaces (e.g. membranes, glass slides)

fixed samples are “probed” with a nucleic acid sequence of interest

hybridization kinetics are very similar to that of nucleic acids in solution

DNA labeling

attachment of radioactive, fluorescent, or other type of marker to DNA molecules

numerous methods exist, including random primer labeling

random primer labeling

a labeling technique that uses complex mixture of random oligonucleotides (usually hexamers) as primers to initiate DNA synthesis

“Klenow fragment” of E. coli DNA pol I is used, together with “labeled” dNTPs (this fragment is the specific enzyme domain with polymerase and 3’-to-5’ exonuclease activities")

radioactive precursors are incorporated into DNA in a random fashion

random primer labeling steps

denature DNA to single strands

anneal hexanucleotide random primer

extend primers with Klenow fragment of DNA pol I in presence of radioactive precursor

denature to single strands to use as probes

detection of labeled molecules- radioactive

32P or 33P, 35S, 3H

detected with x-ray sensitive film (autoradiography) or a radiation-sensitive phosphorescent screen (phosphorimaging)

non-radioactive labeling molecules

fluorescence and chemiluminescence

non-radioactive fluorescence labeling

molecules labeled with fluorophores (dyes) with different emission wavelengths, detected with film or fluorescence detector

non-radioactive chemiluminescence labeling

makes use of reaction between label and additional chemicals. reaction generates light, hybridization signal detected with film

e.g. Digoxigenin (DIG)-dUTP

Southern blotting/transfer/hybridization steps

gel electrophoretogram containing DNA sequences of interest

set up, top to bottom: weight, paper towels, nitrocellulose sheet, gel electrophoretogram containing DNA of interest, wick, buffer solution

NaPH denature and blot onto nitrocellulose sheet

nitrocellulose replica of the gel electrophoretogram

incubate the nitrocellulose-bound DNA with 32p-labeled DNA or RNA of a specific sequence

autoradiograph

autoradiogram shows lines of DNA complementary to 32p-labeled probe

Northern blotting/transfer/hybridization

RNA extract is electrophoresed under denaturing conditions in an agarose gel

after ethidium bromide staining, two bands are typically seen, these are the large and small subunit rRNA molecules which are highly abundant in most cells. the smaller rRNAs, which are also abundant, are usually not seen because they are so short that they run off the bottom of the gel

in most cases, none of the mRNAs are abundant enough to form visible bands after ethidium bromide staining. a light smear of mRNAs of various sizes can sometimes be seen

the gel is blotted on to a nylon membrane and, in this example, probed with a radioactively labeled DNA fragment. in this example, a single band is visible on the autoradiograph, showing that the DNA fragment used as the probe contains part or all of one expressed sequence

microarrays/ microchip arrays/DNA chips

glass surfaces (typically microscope slides) with thousands of DNA fragments arrayed at discrete sites (“spots”)

DNA spots are hybridized to complex samples of fluorescently labeled DNA or RNA in solution

hybridization signals analyzed/compared

microarray steps

isolate mRNAs from cells at two stages of development; each mRNA sample represents all the genes expressed in the cells at that stage

convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled deoxyribonucleotide triphosphates

add the cDNAs to a microarray; fluorescent cDNAs anneal to complementary sequences on the microarray

each fluorescent spot represents a gene expressed in the cells