Laboratory techniques with potential use for computation

The existing models of DNA computation are based on various combinations of the following primitive bio-operations (Thanks to Luiz Merkle for the graphical illustration of bio-operations. The graphical symbols used can be found clicking here. )

-- Synthesizing a desired polynomial--length strand, used in all models. In standard solid-phase DNA synthesis, a desired DNA molecule is built up nucleotide by nucleotide on a support particle in sequential coupling steps. For example, the first nucleotide (monomer), say A, is bound to a glass support. A solution containing C is poured in, and the A reacts with the C to form a two-nucleotide (2-mer) chain AC. After washing the excess C solution away, one could have the C from the chain AC coupled with T to form a 3-mer chain (still attached to the surface) and so on.

-- Mixing: pour the contents of two test tubes into a third one to achieve union. Mixing can be performed by rehydrating the tube contents (if not already in solution) and then combining the fluids together into a new tube, by pouring and pumping for example.

-- Annealing : bond together two single-stranded complementary DNA sequences by cooling the solution. Annealing in vitro is also known as hybridization.

-- Melting : break apart a double-stranded DNA into its single-stranded complementary components by heating the solution. Melting in vitro is also known under the name of denaturation.

-- Amplifying (copying) : make copies of DNA strands by using the Polymerase Chain Reaction (PCR). PCR is an in vitro method that relies on DNA polymerase to quickly amplify specific DNA sequences in a solution. Indeed, the DNA polymerase enzymes perform several functions including replication of DNA. The replication reaction requires a guiding DNA single-strand called template, and a shorter oligonucleotide called a primer, that is annealed to it. Under these conditions, DNA polymerase catalyzes DNA synthesis by successively adding nucleotides to one end of the primer. The primer is thus extended in one direction until the desired strand that starts with the primer and is complementary to the template is obtained. PCR involves a repetitive series of temperature cycles, with each cycle comprising three stages: denaturation of the guiding template DNA to separate its strands, then cooling to allow annealing to the template of the primer oligonucleotides, which are specifically designed to flank the region of DNA of interest, and, finally, extension of the primers by DNA polymerase. Each cycle of the reaction doubles the number of target DNA molecules, the reaction giving thus an exponential growth of their number.

-- Separating the strands by length using a technique called gel electrophoresis that makes possible the separation of strands by length. The molecules are placed at the top of a wet gel, to which an electric field is applied, drawing them to the bottom. Larger molecules travel more slowly through the gel. After a period, the molecules spread out into distinct bands according to size.

-- Extracting those strands that contain a given pattern as a substring by using affinity purification. This process permits single strands containing a given subsequence v to be filtered out from a heterogeneous pool of other strands. After synthesizing strands complementary to v and attaching them to magnetic beads, the heterogeneous solution is passed over the beads. Those strands containing v anneal to the complementary sequence and are retained. Strands not containing v pass through without being retained.

-- Cutting DNA double-strands at specific sites by using commercially available restriction enzymes. One class of enzymes, called restriction endonucleases, will recognize a specific short sequence of DNA, known as a restriction site. Any double-stranded DNA that contains the restriction site within its sequence is cut by the enzyme at that location.

-- Ligating : paste DNA strands with compatible sticky ends by using DNA ligases. Indeed, another enzyme called DNA ligase, will bond together, or ``ligate'', the end of a DNA strand to another strand.

-- Substituting : substitute, insert or delete DNA sequences by using PCR site-specific oligonucleotide mutagenesis. The process is a variation of PCR in which a change in the template can be induced by the process of primer modification. Namely, one can use a primer that is only partially complementary to a template fragment. (The modified primer should contain enough bases complementary to the template to make it anneal despite the mismatch.) After the primer is extended by the polymerase, the newly obtained strand will consist of the complement of the template in which a few oligonucleotides have been ``substituted'' by other, desired ones.

-- Marking single strands by hybridization: complementary sequences are attached to the strands, making them double-stranded. The reverse operation is unmarking of the double-strands by denaturing, that is, by detaching the complementary strands. The marked sequences will be double-stranded while the unmarked ones will be single-stranded.

-- Destroying the marked strands by using exonucleases, or by cutting all the marked strands with a restriction enzyme and removing all the intact strands by gel electrophoresis. (By using enzymes called exonucleases, either double-stranded or single-stranded DNA molecules may be selectively destroyed. The exonucleases chew up DNA molecules from the end inward, and exist with specificity to either single-stranded or double-stranded form.)

-- Detecting and Reading: given the contents of a tube, say ``yes'' if it contains at least one DNA strand, and ``no'' otherwise. PCR may be used to amplify the result and then a process called sequencing is used to actually read the solution. The basic idea of the most widely used sequencing method is to use PCR and gel electrophoresis. Assume we have a homogeneous solution, that is, a solution containing mainly copies of the strand we wish to sequence, and very few contaminants (other strands). For detection of the positions of A 's in the target strand, a blocking agent is used that prevents the templates from being extended beyond A 's during PCR. As a result of this modified PCR, a population of subsequences is obtained, each corresponding to a different occurrence of A in the original strand. Separating them by length using gel electrophoresis will give away the positions where A occurs in the strand. The process can then be repeated for each of C , G and T, to yield the sequence of the strand. Recent methods use four different fluorescent dyes, one for each base, which allows all four bases to be processed simultaneously. As the fluorescent molecules pass a detector near the bottom of the gel, data are output directly to an electronic computer. The bio-operations listed above, and possibly others, will then be used to write ``programs''. A ``program'' will receive a tube containing DNA strands encoding information as input, and return as output either ``yes'' or ``no'' or a set of tubes. A bio-computation will consists of a sequence of bio-operations performed on tubes containing DNA strands.