C value paradox, DBA renaturation kinetics

1. C-VALUE PARADOX,
DNA RENATURATION KINETICS
Submitted by:
Hasniya K.M
Roll. No: 9
1st M.Sc. Botany
St. Teresa’s College, Ernakulam
Submitted to:
Dr. Arya P. Mohan
Asst. Professor
St. Teresa’s College, Ernakulam
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2. Genome
• Genome is the sum of all genetic material in an individual.
• A genome is the complete set of genetic information in an organism.
• It provides all of the information the organism requires to function.
• In living organisms, the genome is stored in long molecules of DNA called
chromosomes. Small sections of DNA, called genes, code for the RNA and
protein molecules required by the organism.
• In eukaryotes, each cell’s genome is contained within a membrane-bound
structure called the nucleus.
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4. • Quantity of DNA in an organism per cell, in all cells, is always constant for a given
species.
• All the organisms on this planet have its own genome whose size varies from one
species to the other and no two species have the same amount of genome nor
the same genomic value or character.
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5. C-value
• C-value or genomic value (Swift-1950) is the total amount of DNA per genome or
haploid set of chromosomes in an organism.
• C-stands for “constant” or “characteristic” to denote that C values are relatively
constant within a single species, but vary widely between species.
• The C-value of a species is usually constant and in general, it increases with
increase in the genetic complexity of species.
• The C-value is expressed as picogram (pg) or base pair (bp).
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6. Examples of C-value
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7. • In the late 1960’s scientists started looking at the complexity of the genome itself. It
was thought that the amount of DNA in a genome correlated with the complexity of
an organism.
• There is linear relationship between C-value and organism complexicity.
• The idea was that the more complex the species the more genes it needed.
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8. • There is a continuous increase in genome size going from a virus to a
mammal this should be expected too.
• Organisms which are more complex need a higher number of genes to
support their complexity. Higher number of genes mean a bigger genome
size.
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9. • However, there are glaring exceptions to the above generalization. Organisms
that are definitely less complex have a bigger genome size as compared to the
organisms that are more complex (some amphibians as compared to human
beings); organisms which have a similar level of complexity have widely differing
genome sizes (housefly vs. fruitfly; one amphibian vs. another).
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10. The exceptions that we have seen above give rise to what we term as the C value
paradox.
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11. C-value paradox
• C-value paradox (Cavalier-Smith 1978) is the paradox that though C-value is an
index of genetic complexity, there is no direct correlation between the
comparative C-values of different species and their relative organizational
complexities and evolutionary status.
• In other words, there is no obvious and direct correlation between different
species of organisms with regard to their relative C-values and organic
complexities.
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12. • There is a steady increase in genome size with increase in the complexity in the
lower eukaryotes.
• There is no good relationship between genome size and morphological
complexity of the higher eukaryotes.
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13. • Some salamanders have more than 30 times the amount of DNA per cell as
humans.
• The C value of human beings is between 3 and 3.4pg, whereas the C-value of
maize is 3.9 pg, and that of frog is 7.6 pg.
• Similarly, human beings have only about 3.3 billion base-pairs in the haploid
genome in place of more than 200 billion base pairs of Amoeba, and over the 300
billion base-pairs of an average bony fish.
• The basic reason for the C-value paradox is that in species with very high C-
values, a large portion of the genomic DNA contains repetative sequence,
pseudogenes, introns and non-coding regions.
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14. Repetative sequence
• Repetitive DNA sequences are a major component of eukaryotic genomes and
may account for up to 90% of the genome size.
• Repeated sequences (also known as repetitive elements, repeating units or
repeats) are short or long patterns of nucleic acids (DNA or RNA) that occur in
multiple copies throughout the genome.
Introns
• Introns are noncoding sections of an RNA transcript, or the DNA encoding it, that
are spliced out before the RNA molecule is translated into a protein.
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15. Non-coding DNA
• Non-coding DNA sequences are components of an organism’s DNA that do not
encode protein sequences. Some non-coding DNA is transcribed into functional
non-coding RNA molecules.
Pseudogenes
• A pseudogene is a segment of DNA that structurally resembles a gene but is not
capable of coding for a protein.
• Pseudogenes are most often derived from genes that have lost their protein-
coding ability due to accumulated mutations that have occurred over the course
of evolution.
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16. DNA RENATURATION KINETICS
Denaturation and renaturation kinetics are used to determine the size and
complexity of the genome. It is also used to understand the relativity of two
genomes and repetitive sequences present in a genome.
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17. Denaturation
• Genomic DNA is first sheared into fragments measuring -1 kb. These double stranded
fragments are now heated. This results in the denaturation (strand separation) of DNA.
• Denaturation can be done by heating (>52°C). The temperature at which DNA is half
denatured is called critical temperature or melting temperature, Tm.
• In the process of denaturation, an unwinding of DNA double-strand takes place,
resulting in two separate single strands.
• It involves breakage of hydrogen bonds between complementary base pairs.
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18. DNA Renaturation
• Separate single strands rewind on cooling and the process is known as
renaturation.
• Renaturation is also known as annealing. When the temperature and pH return to
optimum biological level, the unwound strand of DNA rewind and give back the
dsDNA.
• The renaturation rate is directly proportional to the number of complementary
sequences present.
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19. • After denaturation each strand is randomly distributed in the solution. If
complementary strands have to reassociate then they have to collide with each
other first.
• If each strand has a unique sequence then all the strands will reassociate at about
the same time because all will have the same likelihood of finding their partner
complementary sequences.
• However, if some sequence is repeated, then there will be many fragments
containing these repeated sequences and the likelihood of these repeated
fragments finding each other will be much higher as compared to those sequences
which are represented just once.
• Thus, fragments bearing repeated sequences will reassociate quickly and the
fragments bearing unique sequences will take longer to reassociate.
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20. • The summary of what we have just stated is given below.
1. Single copy DNA will take a long time to reassociate.
2. The more repeated a given sequence is, the quicker it will reassociate.
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21. Let us now see the mathematical part of it.
Renaturation of DNA depends upon random collision of the complementary
strands. This reaction follows a second order kinetics. Therefore the rate of this
reaction will be governed by the equation
DC/dt = -kc²
Where C is the concentration of completely denaturated DNA at time t, and k is the
reassociation rate constant. Integration and some algebraic substitution shows
that,
C/Co = 1/1+kCot
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22. • The above equation tells you one thing very clearly the factor which controls
reassociation reaction is the product of DNA concentration (Co) and incubation
time (t).
At half renaturation,
C/Co = 0.5 (at t = t ½ )
Now,
0.5 = 1 / 1+kCot ½
1+kCot ½ = 1/0.5
1+kCot ½ = 2
kCot ½ = 1
Cot ½ = 1/k
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23. Analysis
1. During reassociation ssDNA find it’s complementary, so that means common
sequence renature more faster than rare sequence. Reassociation kinetics is
faster in repetative DNA
2. Concentration is inversely proportional to rate constant.
Greater Cot ½ → Slower reaction
Lesser Cot ½ → Faster reaction
Cot analysis is a biochemical technique that is used to find out the measure of
repetitive DNA in a DNA sample.
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24. REFERENCE
1. Cooper, G., & Hausman, R. (2013). The Cell: A Molecular Approach. Sunderland:
Sinauer Associates.
2. Karp. G. (2013). Cell Biology. New Jersey: Wiley.
3. Upadhyay, A., & Upadhyay, K. (2005). Basic Molecular Biology. India: Himalaya
Publishing House.
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25. Thank you