DNA re-association kinetics
What is Reassociation of DNA?
Reassociation is the recombination of the products of dissociation. It is very
common in molecular microbiology. The term is most commonly used in DNA
amplification, the process by which DNA is copied in the lab.
What do you mean by reassociation kinetics?
A technique that measures the rate of reassociation of complementary strands of
DNA derived from a single source.
The DNA under study is fragmented into pieces several hundred base pairs in
length and then disassociated into single strands by heating. Subsequently, the
temperature is lowered and the rate of reannealing (q.v.) is monitored.
Reassociation of DNA is followed in the form of a cot curve, which plots the
fraction of molecules that have reannealed against the log of cot. Cot values are
defined as C0 × t, where C0 is the initial concentration of single-stranded DNA
in moles of nucleotides per liter and t is the reannealing time in seconds.
If a DNA molecule is melted and allowed to reassociate, the complexity of the
genome dictates the rate in which duplex DNA will form. If we consider a
simple molecule that consists of alternating GCs, this molecule will be able to
form a duplex quicker than a molecule that consists of repeating blocks of
AGCT.
In practice, the optimum temperature for reassociation is 25°C below the
melting temperature (Tm), that is, the temperature required to dissociate 50%
of the duplex.
What are repetitive sequences in the genome?
The term “repetitive sequences” refers to homologous DNA fragments that
are present in multiple copies in the genome. Repetitive DNA sequences are
present in all higher plants and can account for up to 90% of the genome size in
some species.
In many organisms, a significant fraction of the genomic DNA is repetitive,
with over two-thirds of the sequence consisting of repetitive elements in
humans. Some of these repeated sequences are necessary for maintaining
important genome structures such as telomeres or centromeres.
Repeated sequences are categorized into different classes depending on features
such as structure, length, location, origin, and mode of multiplication.
While some repeated DNA sequences are important for cellular functioning and
genome maintenance, other repetitive sequences can be harmful. Many
repetitive DNA sequences have been linked to human diseases such as
Huntington's disease and Friedreich's ataxia.
1. Tandem Repeats
Tandem repeats are repeated sequences which are directly adjacent to each
other in the genome. Tandem repeats may vary in the number of nucleotides
comprising the repeated sequence, as well as the number of times the sequence
repeats. When the repeating sequence is only 2-10 nucleotides long, the repeat
is referred to as a short tandem repeat (STR) or microsatellite. When the
repeating sequence is 10-60 nucleotides long, the repeat is referred to as
a minisatellite. For minisatellites and microsatellites, the number of times the
sequence repeats at a single locus can range from twice to hundreds of times.
2. Interspersed Repeats
Interspersed repeats are identical or similar DNA sequences which are found in
different locations throughout the genome. Interspersed repeats are
distinguished from tandem repeats in that the repeated sequences are not
directly adjacent to each other but instead may be scattered among different
chromosomes or far apart on the same chromosome.
3. Direct and Inverted Repeats
While tandem and interspersed repeats are distinguished based on their location
in the genome, direct and inverted repeats are distinguished based on the
ordering of the nucleotide bases. Direct repeats occur when a nucleotide
sequence is repeated with the same directionality. Inverted repeats occur when a
nucleotide sequence is repeated in the inverse direction. For example, a direct
repeat of "CATCAT" would be another repetition of "CATCAT." In contrast,
the inverted repeated would be "TACTAC." Repeat CATCAT
When there are no nucleotides separating the inverted repeat, such as
"CATCATTACTAC," the sequence is called a palindromic repeat.
