Thursday, December 6, 2012

Cot Value Analysis

Hi,
Wish you a good time :) Molecular biology! I was taught about "cot value analysis" in my molecular biology class. Here, you could read something, which I know about cot value.

Cot (C not (zero) t) value analysis:
This analysis uses the DNA denaturation and renaturation kinetics. This is used for measuring the repetitive DNA sequence in a given DNA sample. This analysis has application like separating repetitive sequences from mixture of DNA sample. Okay, let us discuss the procedure now.

Note:
Cot value = (C zero) * (Time) * (constant representing the effect of cation in the buffer on renaturation)
where,
c zero -> DNA concentration (mol/l)
T - Renaturation time (sec)

Procedure:
  1. Denature the DNA sample by heating
  2. Cool and renature the sample, note renaturing extent (as cot value) at particular temperatures until a particular cot value is reached.
  3. Now, dilute this sample with sodium phosphate buffer- SPB (0.03M - at this buffer concentration the DNA bind to hydroxyl apatite properly)
  4. Bind this buffer solution with DNA to hydroxylapatite(HAP) column.
  5. 0.12M SPB is now used for eluting Single stranded DNA from HAP; Collect the Single stranded DNA.
  6. 0.50M SPB is then used for eluting double stranded DNA which is also collected.
  7. Volumes of Single and double stranded DNA collected is measured.
  8. 0.9ml of the centrifuged single stranded DNA is mixed with 0.1 ml of  10N KOH. This addition of KOH denatures the double stranded DNA (if present). Do this to the double stranded DNA sample also.
  9. Absorption values A260 (adjusted for light scatter at 360nm) are obtained for both the samples prepared (mentioned in the previous point)
Percentage of single stranded DNA could be calculated with the help of the following formula:

[(Vss x Ass) x 100] ÷ [(Vss x Ass) + (Vds x Ads)] = % ssDNA
where,
Vss = total volume of single-strand fraction, 
Vds = total volume of double-strand fraction, 
Ass = A260(adjusted for light scatter) for the KOH-denatured single-strand fraction,
Ads = A260 (adjusted for light scatter) for the KOH denatured double-strand fraction.

Background:
  • The rate of renaturation of repetitive sequences will be high comparing the rate of renaturation of non repetitive or less repetitive sequences.
  • This is because, the possibility of a repetitive sequence to find it's complementary strand is greater than that of a non repetitive strand. (Hope you got it)
For detailed explanation refer "here"

Got any doubts? Kindly comment I'll try to answer.

Bye, Take CARE! :) 



2 comments:

  1. Cool and renature the sample, note renaturing extent (as cot value) at particular temperatures until a particular cot value is reached.
    How is this step done?

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    Replies
    1. Cooling can be done by using a water bath, simple? And using the formula for cot value calculation, get the cot values. For detailed explanation refer the following link:

      http://www.mgel.msstate.edu/pdf/cot.pdf

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