2.1 Properties of nucleic acids
Nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are extremely long polymers made up of phosphate-sugar-nitrogenous base (nucleotide) units. The bases found in DNA are adenine and guanine (both purines) and cytidine and thymine (both pyrimidines). Thymine is replaced by uracil in RNA. The nucleotides are linked by 3' to 5' phosphodiester bonds. That is, a phosphate group on the 5' position of the sugar residue becomes linked to the 3' hydroxyl group of the preceding sugar group on the chain as the long polymer is synthesized.
Figure 1. The structure of nucleic acids showing the bases found in DNA and how nucleotides are linked together.
The double stranded structure of DNA was worked out in 1953 by Watson and Crick . It is absolutely essential to understand the key concepts behind this structure since all cloning strategies are based upon them.
The double stranded (double helix) molecule consists of two strands wound around a central axis with the bases stacked inside. The order of the strands are in opposite directions (5' to 3' in one, 3' to 5' in the other). The bases stack together in the center of the helix because they interact with one another via weak hydrogen bonds.
Figure 2. Structural features of the DNA double helix.
Hydrogen bonds are much weaker than covalent bonds and are continually forming and disassociating. They form between two electronegative atoms, one of which has a covalent hydrogen. The special properties of water are due to hydrogen bonds of the type:
In double stranded nucleic acids, adenine will only form hydrogen bonds with thymine (or uracil) and cytidine will only form hydrogen bonds with guanine. Thus, the amount of adenine always equals the amount of thymine and the amount of cytidine always equals that of guanine for the DNA of a given species. The per cent G-C (and thus the per cent A-T) in DNA is surprisingly variable from species to species.
Figure 3. Base pairing between A:T and G:C.
The base pairs form a flat plane in the interior of the helix with adenine forming two hydrogen bonds with thymidine and cytidine forming three hydrogen bonds with guanine. It immediately occurred to Watson and Crick that if you zipped open the two strands and began copying them so that everywhere that there was an adenine in the original strand, the new strand had a thymidine, (a guanine would be matched to a cytidine etc.), then soon you would exactly duplicate both strands using only the concept of base pairing and whatever enzymes and substrates that might be necessary to carry out the synthesis.
At the completion of replication, each original strand (parental strand) is paired with a new (daughter) strand. The replication is said to be semiconservative. Double stranded DNA may be denatured by alkaline conditions or by heat. For example, the two strands will unwind when a solution of DNA is heated to 90o and then will reassociate if the temperature is lowered gradually. Longer DNA molecules take longer to reform their double-stranded structure in perfect alignment.
Even small stretches of DNA (polynucleotides) will anneal with large single-stranded DNA molecules if the base sequence matches a sequence somewhere on the single stranded DNA (by base pairing). A sequence of 10 or more matching base pairs usually is needed to form enough hydrogen bonds for the complex to be stable. Many of techniques used to manipulate DNA make use of this observation.