Mutation detection and identification

#1. Field

#The present teachings generally relate to nucleic acid analysis, and in various embodiments, to a system and methods for detecting and identifying heterozygous indel mutations.

#2. Description of the Related Art

#These and other aspects, advantages, and novel features of the present teachings will become apparent upon reading the following detailed description and upon reference to the accompanying drawings. In the drawings, similar elements have similar reference numerals.

#FIGS. 1A, B, C illustrate exemplary sequence traces associated with mixed-base analysis.

#FIG. 2 illustrates exemplary sequence traces for a sample containing multiple alleles.

#Reference will now be made to the drawings wherein like numerals refer to like elements throughout. As used herein, “target”, “target polynucleotide”, “target sequence” and “target base sequence” and the like refer to a specific polynucleotide sequence that may be subjected to any of a number of sequencing methods used to determine its composition (e.g. sequence). The target sequence may be composed of DNA, RNA, analogs thereof, or combinations thereof. The target may further be single-stranded or double-stranded. In sequencing processes, the target polynucleotide that forms a hybridization duplex with a sequencing primer may also be referred to as a “template”. A template serves as a pattern for the synthesis of a complementary polynucleotide (Concise Dictionary of Biomedicine and Molecular Biology, (1996) CPL Scientific Publishing Services, CRC Press, Newbury, UK). The target sequence may be derived from any living or once living organism, including but not limited to prokaryote, eukaryote, plant, animal, and virus, as well as synthetic and/or recombinant target sequences.

#Furthermore, as used herein, “sample assembly” and “assembly” refer to the reassembly or consensus analysis of smaller nucleotide sequences or fragments, arising from individually sequenced samples that may comprise at least a portion of a target sequence. By combining the information obtained from these fragments a “consensus sequence” may be identified that reflects the experimentally determined composition of the target sequence.

#Nucleic acid sequencing, according to the present teachings, may be performed using enzymatic dideoxy chain-termination methods. Briefly described, these methods utilize oligonucleotide primers complementary to sites on a target sequence of interest. For each of the four possible bases (adenine, guanine, cytosine, thymine), a mixed population of labeled fragments complementary to a least a portion of the target sequence may be generated by enzymatic extension of the primer. The fragments contained in each population may then be separated by relative size using electrophoretic methods, such as gel or capillary electrophoresis, to generate a characteristic pattern or trace. Using knowledge of the terminal base composition of the oligonucleotide primers along with the trace information generated for each reaction allows for the sequence of the target to be deduced. For a more detailed description of sequencing methodologies the reader is referred to DNA sequencing with chain-terminating inhibitors, Sanger et. al., (1977) and A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides, Prober et al., (1987).