Non-electrophoretic DNA sequencing methods

A few DNA sequencing methods which are not electrophoretic based have been conceived in the last decades. These techniques have advantages and disadvantages compared to electrophoretic separation methods, depending on the type of applications performed and have promising applicability into miniaturized systems.

Sequencing-by-hybridization

The sequencing-by-hybridization (SBH) technique is in fact based on the same principle as that of Southern blotting [51] and was simultaneously described by two independent research groups in 1988 [52]. It key premise was its ability to facilitate de novo sequencing with the development of hybridization arrays[53-54]. The technique entails the labeling and subsequent annealing of an unknown strand of DNA to an array of short length oligonucleotides (e.g. all

65,536 combinations of 8-mers) which is followed by the analysis of the sequence by the pattern formed from its hybridization with the array. For this a computer assisted assembly program is essential for the ultimate reconstruction of the correct order and sequence of the original strand (Figure 8).

Because of the sheer scale of the data set from which the sequence is determined, the SBH technique has been prone to produce ambiguous results due to repetitive regions within the unknown sequence. Additionally, the formation of secondary structures within the target also reduces the accuracy of this technique. False positives are also caused by single base mismatches resulting from variations within the stability of the duplex of a complete match; this mostly occurs at the 3’-terminus. The effect of these issues can be minimized with the use of expression analysis or comparative sequencing between SBH microarrays though these issues have still not been solved in de

novo sequencing.

Figure 8 Sequencing-by-hybridization (SBH) (i.e. the reverse dot-blot format).

An unknown target is labeled and hybridized with an array of known octamer oligonucleotides, with the formed hybridization pattern being interpreted to determine the sequence of the targeted DNA sequence.

Sequencing-by-synthesis

The approach referred to as sequencing-by-synthesis (SBS) was first described in 1985 by Robert Melamede [55]. This technique consists of the

sequential addition and incorporation of nucleotides in an extension reaction directed by a polymerase primer in such a manner that a strand complementary to the original forms through interactive addition of the four dNTPs. Figure 9 shows the principle of SBH.

The detection of incorporation of nucleotides in the primed DNA strand can be done either directly or indirectly. In the direct method, fluorescent labels are attached to the dNTPs to allow their detection with an appropriate detection method [56-57]. A draw back to this technique is the low number of bases that can be added due to the high failure rate of nucleotide incorporation which can in turn lead to desynchronization of the sequence being copied [57]. The post- synthesis removal of the fluorophore acts as one further step in the process during which errors can be introduced.

The indirect approach to detection employs natural nucleotides for the incorporation process. The pyrophosphate (PPi) released during the condensation polymerization reaction of the growing chain is then detected as an indirect indication of successful nucleotide incorporation [58-59]. The principal advantage of this approach is the ability to use unadulterated, naturally occurring nucleotides which in turn exhibits superior incorporation efficiency in the assay. The enzymatic process laid out in the scheme below details is based on the luminometric detection of ATP through the conversion of PPi molecules through an enzymatic cascade.

(DNA) + dNTP!"#$%&'()*,-.*/000000000001 2DNA3 45+ $PPi$ (1)

PPi$ + $APS#67$.8'%98,('-.*/0000000000001 ATP$ + $ SO:;< (2)

ATP$ + $luciferin$ + $ O<

'8=>?*,-.*

Figure 9. An illustration of the underlying principal of sequencing by synthesis

consisting on the primed DNA template being subjected to iterative additions of nucleotides while in the vicinity of a DNA polymerase molecule, represented in the schematic as an oval shape. If a complimentary nucleotide is added, incorporation of it into the sequence can be conducted through a condensation reaction, releasing a PPi molecule and polymerizing the nucleotide to the chain.

With the addition of dNTP to the reaction mixture, the DNA polymerase incorporates a complimentary nucleotide onto the 3’terminus of the strand which releases a PPi molecule into the solution. The free PPi molecule can then be observed via a coupled enzymatic reaction consisting of the ATP sulfurylase- mediated conversion of PPi to ATP which is in turn consumed by the firefly luciferase enzyme to produce detectable fluorescence [58, 60]. Detection of the signal is done with a photon multiplier or a charge coupled device (CCD) camera. This method does not, however, lend itself to the robust sequencing of DNA and thus no further progression of the technique is reported in the literature.

In progressive stages over the course of time, the sequencing-by- synthesis method has been developed into a robust, easy-to-use sequencing method for DNA through improvements in the removal efficiency of nucleotides and additional modification of the sequencing-by-synthesis principal. The innovation of the Pyrosequencing technology developed and described by Nyrén and colleagues is a direct result of this [61-63].