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    Cot Curves

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    Venkateswarlu Yadavalli
    The document discusses denaturation and renaturation of nucleic acid polymers. It explains how spectrophotometry can be used to measure the relative amounts of single- and double-stranded DNA by monitoring absorbance over a temperature range. The temperature at which 50% of the DNA is denatured is called the transition temperature or Tm. The Tm is affected by base composition, duplex length, and ionic strength of the solution. Renaturation is the reverse process of denaturation and is affected by temperature, ionic strength, DNA concentration, denaturing agents, and mismatches between strands. Cot curves plot percent renaturation over time and concentration to measure sequence complexity.

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    Cot Curves

    Uploaded by

    Venkateswarlu Yadavalli
    5K views4 pages
    The document discusses denaturation and renaturation of nucleic acid polymers. It explains how spectrophotometry can be used to measure the relative amounts of single- and double-stranded DNA by monitoring absorbance over a temperature range. The temperature at which 50% of the DNA is denatured is called the transition temperature or Tm. The Tm is affected by base composition, duplex length, and ionic strength of the solution. Renaturation is the reverse process of denaturation and is affected by temperature, ionic strength, DNA concentration, denaturing agents, and mismatches between strands. Cot curves plot percent renaturation over time and concentration to measure sequence complexity.

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    The document discusses denaturation and renaturation of nucleic acid polymers. It explains how spectrophotometry can be used to measure the relative amounts of single- and double-stranded DNA by monitoring absorbance over a temperature range. The temperature at which 50% of the DNA is denatured is called the transition temperature or Tm. The Tm is affected by base composition, duplex length, and ionic strength of the solution. Renaturation is the reverse process of denaturation and is affected by temperature, ionic strength, DNA concentration, denaturing agents, and mismatches between strands. Cot curves plot percent renaturation over time and concentration to measure sequence complexity.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    5K views4 pages

    Cot Curves

    Uploaded by

    Venkateswarlu Yadavalli
    The document discusses denaturation and renaturation of nucleic acid polymers. It explains how spectrophotometry can be used to measure the relative amounts of single- and double-stranded DNA by monitoring absorbance over a temperature range. The temperature at which 50% of the DNA is denatured is called the transition temperature or Tm. The Tm is affected by base composition, duplex length, and ionic strength of the solution. Renaturation is the reverse process of denaturation and is affected by temperature, ionic strength, DNA concentration, denaturing agents, and mismatches between strands. Cot curves plot percent renaturation over time and concentration to measure sequence complexity.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 4

    adapted from Roger Meisfeld, University of Arizona

    Denaturation and Renaturation of Nucleic Acid Polymers The relative amount of single- or double-stranded DNA in solution can be experimentally determined using spectrophotometery to measure ultraviolet light absorbance at a wavelength of 260 nanometers (OD260). The aromatic bases in DNA are less accessible to ultraviolet light in the double-stranded, compared to singlestranded form, which creates a measurable difference in the observed OD260. Using this empirical difference in absorbance, it is possible to observe the effect of temperature on DNA structure by monitoring OD260 over a range of temperature range. The temperature at which 50% of the DNA is denatured is called the Tm or transition temperature.

    adapted from Roger Meisfeld, University of Arizona

    The Transition Temperature (Tm) of a nucleic acid duplex is strongly effected by three factors; 1) Base composition 2) Duplex length 3) Ionic strength of the solution Two molecular genetic applications where extent of duplex formation is an important consideration are the use of short oligonucleotides in hybridization reactions, and heteroduplex formations between molecules that are less than 100% complementary. The use of homologous, but not identical, DNA molecules in hybridization reactions is common when sequence divergence exists between two genes, for example, across species or among members of a related gene family. The reverse of nucleic acid denaturation is renaturation, also referred to as reassociation or hybridization. This bimolecular process is most effected by - temperature - ionic strength, - molar concentration of the two complementary strands - reaction time - denaturing agents such as formamide or urea which lower the Tm - dextran sulfate which increases the rate of reassociation

    adapted from Roger Meisfeld, University of Arizona

    Cot Curves The term Cot is used to describe the kinetics of hybridization between two nucleic strands in solution and is defined by the product of [nucleic acid] x (time). Put simply, when the concentration of two complementary strands in a solution is high, then it takes a shorter time for hybridization to occur than it does when one or both of the strands are present at a low concentration. Cot curves plot percent reassociation versus Cot (mole secs/liter), and are used to measure the sequence complexity of DNA samples . DNA from organisms with small genomes have low sequence complexity and reassociate at much lower Cot values, than do denatured DNA samples from more complex organisms.

    adapted from Roger Meisfeld, University of Arizona

    Factors that effect the denaturation and renaturation of nucleic acid duplexes.

    Parameter

    Effect on Tm

    Effect on rate of renaturation

    base composition

    increase Tm with increase %GC

    no effect

    hybrid length

    <150 bp; increase Tm with increase length >500 bp; no effect

    increase rate with increase length

    ionic strength

    increase Tm with increase [Na ]


    +

    optimal at 1.5M Na

    %bp mismatch

    decrease Tm with increase %mismatch

    decrease rate with increase %mismatch increase rate with increase [DNA]

    DNA concentration

    no effect

    denaturing agents

    decrease Tm with increase [formamide], [urea]

    optimal at 50% formamide

    temperature

    not applicable

    optimal at 20C below Tm

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