Introduction to Recombinant Genetics- Biology 350
Double-stranded DNA can be denatured by heat or strong base. Under the proper conditions of temperature (Tm -20°C) and salt, the complementary strands of DNA can reanneal into their double-stranded state.
Hybridization can occur when DNA from two sources are denatured and then mixed under reannealing conditions. Hybrids between the DNA from the two sources can form in regions of sequence conservation.
Hybridization and reannealing of DNA and/or RNA is concentration and time dependent.
As illustrated in the reannealing curves below, the annealing reactions can be monitored by following the Absorbance of the DNA at 260 nm (remember that ssDNA absorbs about 38% more/mole than does dsDNA). Since the rate of annealing is concentration dependent (the higher the concentration of complementary strands the faster a DNA will find a mate), and time dependent, hybridization and reannealing curves can be made comparable by plotting the Absorbance vs the Cot, where Cot is the initial concentration of the DNA,Co, times the time (min).
In the plot below, the E. coli DNA sample reannealed within in 2 orders of Cot. This is typical of a pure, unrepeated DNA. The dark plot shows several reannealing steps. This is typical of DNA samples that have repeated sequences where those sequences that are most highly repeated, hr, are able to find their complement faster because there is an effectively higher concentration than the rest of the genomic DNA. The mr fraction represents middle repetitive DNA and the nr fraction represents non-repetitive DNA.
From hybridization curves, the number of repeats for each fraction can be determined by comparing the Cot1/2, which is the Cot value read of the X axis at the point that half of the fraction is renatured, between the repetitive and non-repetitive fractions. For example, the number of repeat in the hr fraction can be estimated by taking the ratio of the Cot1/2 for nr and dividing it by the Cot1/2 for hr:
Repeats for hr = 10^2 / 10^-2 = 10^4.
Therefore, hr is repeated 10,000 times more than nr in the genome.
The length of the hr repeat can be estimated by comparison of the Cot1/2 for hr to that of E. coli, where the length of E. coli is known to be 4.2 x 10^6 bp. :
bp for hr = (Cot1/2 hr * f for hr) (4.2 x 10^6 bp) / (Cot1/2 E.coli * f for E.coli )
bp for hr = (10^-2 * 0.3) (4.2 x 10^6 bp) / (10^1 * 1.0) = 1260 bp
Therfore, hr is a 1260 bp repeat that has 10,000 copies in the genome.
You can see the calculations for the other fractions, mr and nr in the figure and table below.
You can also estimate the genome size by multiplying the complexity (complexity is the number of bp in a haploid genome) times the repetitive value for each of the components, nr, mr, and hr and then summing all three products together to get the total bp in the genome.
This example demonstrates the analytical power of hybridization and reannealing kinetics. Such techniques can be extended to estimate the similarity between organisms and to estimate the amount of the genome expressed as RNA.
The most common use of hybridization is not as a quantitative tool but as a qualitative or identification tool. Often, a mixture of ligated fragments are transformed into a host cell and many colony or plaque clones will be generated. Selection and indicator methods, such as blue / white colony screening for lac Z gene products, can be used to distinguish between recombinant and non-recombinant clones. But selecting a recombinant of interest from a mixture of recombinants requires additional techniques such as colony or plaque screening.
Colony or plaque screens begin by lifting part of the colony or plaque from the plate by placing a nitrocellulose or nylon membrane onto the plate and then removing it. The filters are then floated on a solution of alkali that will rupture the cells and denature the DNA. Single-stranded DNA will bind to the filter, allowing the other cellular contents to be washed off. The DNA is then permanently fixed to the filter by heating to 80°C (nitrocellulose) or UV crosslinking (nylon) the DNA to the filter. The filter with the bound ssDNA can then be hybridized to labeled probes specific for the DNA of interest. The clones on the original plate that correspond to the locations on the filter that are bound by the probe can then be selected as the recombinants of interest.
Probes can be labeled by filling in the ends of restriction enzyme sites with labeled nucleotides or end labeling the 3' or 5' ends (see enzyme lecture).
A polular method is to use nick translation or random priming and then extending the nicks or random primers with labeled nucleotides.
Labeled nucleotides can be radioactively tagged or can be tagged with biotin or with a small enzyme such as horseradish peroxidase.
Probes can also be made from RNA and can be generated as run-off transcripts labeled with tagged NTPs.
The temperature of hybidization is dependent on salt concentration and GC content of the probe.
The temperature of hybridization is Th = Tm - 20°C.
The Tm for DNA-DNA hybridization is calculated as follows:
Tm = 81.5°C + 16.6 Log[Na+] + 0.41(%G+C) - 0.61(%formamide) - 500/#bp in duplex
The Tm for RNA probes and DNA targets is calculated as:
Tm = 79.8 °C + 18.5 Log[Na+] + 0.584(%G+C) - 0.5(%formamide) - 820/#bp in duplex
The temperature of hybridization of short oligonucleotide probes is estimated as:
Th = Tm - 5°C = 2°C(#A-T bp) + 4°C(#G-C bp) - 5°C
After hybridization, the membrane is washed with higher stringency, or at higher temperatures up to within 5°C of the Tm to remove non-complementary hybrids. The ionic strangth of the solution and the SDS concentration may also be varied to optimize the signal-to-noise ratio.
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