BIO 101

DNA DENATURATION AND

RENATURATION
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Denaturation
• Process of strand separation
• Involves disruption of hydrogen bonds between
base pairs
• Agents of DNA denaturation
• Heat
• Alkali
• Melting proteins
• eg. RNA polymerase, helicase

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Melting Curve and Tm
• Melting curve
• Plot of extent of denaturation vs. temperature
• Melting temperature (Tm)
• Midpoint of the melting curve
• Temp at which half of the DNA is denatured

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Melting Curve

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Factors Influencing Tm
• Size and base composition of the DNA
• High GC content, high Tm
• Shorter DNA, low Tm
• the conditions employed for denaturation
• ionic strength, pH
• urea, methanol, etc.

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Monitoring Denaturation
• Monitor A260 with increasing temperature
• A260 is expected to increase with increasing temp
• As DNA gets denatured, nitrogenous bases
become more exposed and are able to strongly
absorb UV light
• Hyperchromic shift

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DNA Breathing
• the transient denaturation
• small fraction of base pairs in the genome
• occurs more frequently between bases with fewer H-
bonds
• necessary for gene regulation
• requires no molecules for the event to occur

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DNA Renaturation
• Ability of the two separated complementary strands
to reform into the ordered state
• i.e. double helix
• Two steps
• nucleation
• zippering
• Upon renaturation, A260 decreases
• Hypochromic shift

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DNA Renaturation

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Cot Plot
• Co - initial ssDNA concentration (in moles nucleotide
per liter)
• t - time for renaturation to be completed
(in secs)
• From an analysis of a Cot plot, one can determine:
• genome size
• genome complexity - relative proportions of
single-copy and repetitive sequences

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Cot Plot

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Cot Plot

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Classes of Eukaryotic DNA
based on sequence complexity
• Unique sequence
• 1 to 3 copies per genome
• single copy, low copy, non rep. DNA
• e.g. most coding genes, regulatory sequences

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Classes of Eukaryotic DNA
based on sequence complexity
• Moderately repetitive DNA
• 10 to 10,000 copies per genome
• generally dispersed repeats
• occasionally clustered (i.e. gene families)
• e.g. rRNA and tRNA genes, histone genes

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Classes of Eukaryotic DNA
based on sequence complexity
• Highly repetitive DNA
• 100,000 to 1 M copies per genome
• each copy can range from few nucleotides to
several hundreds
• generally found as tandem repeats
• e.g. centromeric DNA, telomeres DNA, satellite
DNA, some transposons

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 APPLICATIONS OF
DENATURATION/RENATURATION
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1.) Studying Genome
Relatedness
• denatured DNA of two different organisms are
allowed to renature
• percent homology is determined
• reflects the similarity in the sequence of the
various DNA molecules in the genomes of the
species being compared

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1.) Studying Genome
Relatedness
Organism A DNA Organism B DNA

ss DNA A ss DNA B

Mixed under renaturation conditions

DNA A = DNA B hybrid

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2.) RNA Looping
• to detect introns within the cloned gene

mRNA + corresp. gene

hybridize & view
under EM

Intron loop

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2.) RNA Looping

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3.) Probing
• Probe
• ssDNA or RNA fragment (15 – 1000 nt) of known
sequence
• complementary to the target DNA/RNA
• labeled radioactively or non-radioactively
• for detection of specific nucleotide sequences
in the DNA/RNA sample

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Southern Blotting + Hybridization
• Devised by Edward Southern in 1975
• DNA fragments separated on a gel
• Blotted to a charged surface / membrane (e.g.
nitrocellulose membrane)
• hybridized with a DNA probe

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Southern Blotting + Hybridization
• Location of DNA + probe on the filter are detected by
autoradiography
• High sensitivity:
• can detect ~ 5 μg single copy sequence
• Applications:
• RFLP Mapping
• Forensic Investigation
• Genetic Screening

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Southern Blotting + Hybridization
1. Restriction digestion

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Southern Blotting + Hybridization
2. Electrophoresis

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Southern Blotting + Hybridization
3. Blotting, Alkali Denaturation and Fixing

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Southern Blotting + Hybridization
4. Hybridization with radioactive probe

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Southern Blotting + Hybridization
5. Autoradiography

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Other Blotting Techniques
• Northern Blotting
• RNA blotted on membrane
• DNA probe
• Western Blotting
• proteins blotted
• labeled antibody specific for the protein
• Eastern Blotting
• Used to detect glycoproteins and lipoproteins

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