Sachadyn P, Kur J
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
J Theor Biol. 1999 Apr 7;197(3):393-401. doi: 10.1006/jtbi.1998.0880.
The application of degenerate oligonucleotides to DNA Sequencing by Hybridisation with Oligonucleotide Matrix (SHOM) is proposed. The use of degenerate oligonucleotides is regarded as an example of pooling methods that are suitable for various laboratory procedures requiring numerous samples to be assayed. As each DNA sequence coded by four letters (A, G, C, T) may be defined by two sequences: a sequence coded by W and S (W-weak-A or T, S-strong-G or C) and a sequence coded by R and Y (R-purine-A or G, Y-pirymidine-T or C), n4n -nucleotide sequences may be defined with the help of 2xn2sequences. In the place of the originally described microchip matrix composed of all possible unambiguous octanucleotides (4(8)=65 536) attached to the equal number of 65 536 microlocations a matrix composed of 512 microlocations containing 256 2(8)-degenerate octanucleotides is proposed. The matrix contains all 256 possible octanucleotides coded by W and S variations and all 256 possible octanucleotides coded by R and Y variations. The 512 256-degenerate octanucleotides allows to retrieve the same information as 65 536 unambiguous octanucleotides. A variant of the DNA sequence reconstruction method applicable to this system is presented. The use of degenerate oligonucleotides also gives the possibility to apply matrices composed of longer oligonucleotides without increasing the number of microlocations in matrices, which would enable increasing the length of unambiguously reconstructed sequence, e.g. a matrix comprising 131 072 16-mer oligonucleotides i.e. 65 536 65 536-fold degenerate oligonucleotide coded by W and S variations and 65 536 65 536-fold degenerate oligonucleotide coded by R and Y variations could replace one matrix comprising all possible unambiguous 16-mer oligonucleotides (ca. 4.3x10(9)).
提出了将简并寡核苷酸应用于通过与寡核苷酸矩阵杂交进行DNA测序（SHOM）。使用简并寡核苷酸被视为合并方法的一个示例，该方法适用于需要检测大量样品的各种实验室程序。由于由四个字母（A、G、C、T）编码的每个DNA序列可以由两个序列定义：一个由W和S编码的序列（W-弱-A或T，S-强-G或C）和一个由R和Y编码的序列（R-嘌呤-A或G，Y-嘧啶-T或C），借助2×n²个序列可以定义n⁴个n核苷酸序列。代替最初描述的由附着在相等数量的65536个微位置上的所有可能的明确八核苷酸（4⁸ = 65536）组成的微芯片矩阵，提出了一种由512个微位置组成的矩阵，其中包含256个2⁸简并八核苷酸。该矩阵包含由W和S变体编码的所有256种可能的八核苷酸以及由R和Y变体编码的所有256种可能的八核苷酸。这512个256简并八核苷酸能够检索与65536个明确八核苷酸相同的信息。提出了一种适用于该系统的DNA序列重建方法的变体。使用简并寡核苷酸还使得有可能应用由更长的寡核苷酸组成的矩阵，而不增加矩阵中微位置的数量，这将能够增加明确重建序列的长度，例如，一个由131072个16聚体寡核苷酸组成的矩阵，即由W和S变体编码的65536个65536倍简并寡核苷酸和由R和Y变体编码的65536个65536倍简并寡核苷酸，可以替代一个由所有可能的明确16聚体寡核苷酸（约4.3×10⁹）组成的矩阵。
