(11)
7. Definere begreberne DNA denaturering (melting), renaturering (re-annealing)
og hybridisering af prober
Stryer, s.124-5
Devlin, s.42 - s.44, fig.2.20
DNA denaturizing (melting)
- disruption of the hydrogen bonds between the bases in the DNA sequence. The
secondary structure -
the DNA
double helix
is lost.
During DNA replication and other processes, the two strands of the double helix
must be separated from one another, at least in a local region.
This
can also be accomplished in laboratory conditions by heating up the DNA. The
heating disrupts the hydrogen bonds between
the
base pairs, and
thereby causes the strands to separate.
Inside living cells, the double helix is not melted by addition of heat.
Instead, proteins called helicases use chemical energy to disrupt the
structure of the double helix.
Besides the temperature, other factors have also influence on this DNA-melting:
the pH value - at pH of 11.3, the NH- groups of the basic rings become unprotonated (the H-atoms disassociate), thereby loosing their ability to make Watson-Crick bonding
the salt-concentration - a high concentration of catjons, which bind to the negative phosphate backbone, stabilize the DNA structure and decrease the repulsive forces between the two strands. So, lower salt-concentration, bigger denaturizing.
DNA-renaturizing (re-annealing) - if appropriately treated, the separated strands of DNA can re-associate to form a double helix (fx. if the temperature is lowered).
Annealing is possible even after complementary strands have been completely separated. One DNA strand needs to find its complementary strand among a lot of other DNA molecules. In order to find the fitting sequence, the bases form a lot of hydrogen bonds among each other thereby testing if they have found the right strand. The "wrongly" paired strands have a short life, because the bases that lie around the complementary sequence can not pair. Once the correct bases begin to pair by chance, the double helix is rapidly reformed over the entire DNA. The formation of the first base pair is always slow. The second base pair makes hydrogen bonds faster, the third even faster... and so on as the sequence expands.
Hybridization of probes
Hybridization - association between two polynucleotide chains, which may be of same or different origin or length, provided that a base complementarity exists between these chains.
Probe - short single stranded RNA and DNA oligonucleotides (app. 15-20 nucleotides) that are complementary to a specific sequence of interest of the genomic DNA.
The following hybrids can be formed:
DNA-DNA
DNA-RNA
RNA-RNA
The hybridization of probes can be used for several purposes:
location of any given DNA sequence of interest by annealing with a probe, that is appropriately tagged for easy detection of the hybrid (location of genes in DNA that correspond to a particular RNA, by using the RNA as a probe)
DNA molecules from 2 different species can be melted and allowed to hybridize in the presence of each other. The degree of hybridization is an indication of the relatedness of the genomes and hence the organisms.
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