The present invention provides a primer for nucleic acid random fragmentation and a nucleic acid random fragmentation method. The primer consists of a plurality of upstream random primers and downstream random primers. The sequence composition of the upstream random primers is 5′-X-Y-3′, and the sequence composition of the downstream random primers is 5′-P-Y′-X′-close-3′, wherein Y and Y′ are random sequences, X is all or part of sequences of a sequencing platform 5′ end adaptor, X′ is all or part of sequences of a sequencing platform 3′ end adaptor, P is phosphorylation modification, and close is close modification. The primer of the present invention adopts double random anchoring of both the upstream random primers and the downstream random primers, and a DNA sample can be randomly broken.

Detection method of a base mutation in a target base sequence of a nucleic acid sample, includes: performing a PCR reaction with the nucleic acid sample as a template, using a primer set capable of amplifying, by PCR, an amplification target region including the target base sequence; a blocker nucleic acid fragment having a base sequence complementary to the target base sequence and including a residue which is synthetic nucleic acid; and a probe that hybridizes to a region, in the target region, closer to a 5′ end of the target region than the target base sequence in the same chain as the target base sequence, and that has a fluorescent substance on one of a 5′ end and a 3′ end of the probe and a quenching substance on the other; and measuring an amplification amount of the template in the PCR reaction by detecting fluorescence from the probe.

Detection method of a base mutation in a target base sequence of a nucleic acid sample, includes: performing a PCR reaction with the nucleic acid sample as a template, using a primer set capable of amplifying, by PCR, an amplification target region including the target base sequence; a blocker nucleic acid fragment having a base sequence complementary to the target base sequence and including a residue which is synthetic nucleic acid; and a probe that hybridizes to a region, in the target region, closer to a 5′ end of the target region than the target base sequence in the same chain as the target base sequence, and that has a fluorescent substance on one of a 5′ end and a 3′ end of the probe and a quenching substance on the other; and measuring an amplification amount of the template in the PCR reaction by detecting fluorescence from the probe.

Rapid nucleic acid libraries, methods of generation, kits, and compositions relating to library synthesis, including reagents, intermediaries and final products are disclosed herein. The disclosure enables rapid synthesis of libraries that allow independent verification of sequence information and rapid identification of sequence information with template of origin.

Rapid nucleic acid libraries, methods of generation, kits, and compositions relating to library synthesis, including reagents, intermediaries and final products are disclosed herein. The disclosure enables rapid synthesis of libraries that allow independent verification of sequence information and rapid identification of sequence information with template of origin.

Provided herein are systems and methods for nucleic acid sequencing by synthesis in a plurality of wells using detectably labeled chain terminating nucleotides with photolabile blocking groups and pulses of photocleaving light. In certain embodiments, the systems and methods provides a plurality of deblock-scan cycles comprising an initial deblock time period followed by a scanning light period, wherein at least one of the following occurs in each deblock-scan cycle: 1) the deblock time period is shorter than the scan time period; 2) the deblock time period is only long enough to deblock the photolabile groups that are part of a primer in less than all of the plurality of wells; or 3) the deblock time period is between 25 and 150 mSec and the scan time is at least 200 mSec. Such shorter deblock time periods help prevent the addition of more than one nucleotide to the primer prior to scanning (e.g., accuracy is enhanced).

Provided herein are systems and methods for nucleic acid sequencing by synthesis in a plurality of wells using detectably labeled chain terminating nucleotides with photolabile blocking groups and pulses of photocleaving light. In certain embodiments, the systems and methods provides a plurality of deblock-scan cycles comprising an initial deblock time period followed by a scanning light period, wherein at least one of the following occurs in each deblock-scan cycle: 1) the deblock time period is shorter than the scan time period; 2) the deblock time period is only long enough to deblock the photolabile groups that are part of a primer in less than all of the plurality of wells; or 3) the deblock time period is between 25 and 150 mSec and the scan time is at least 200 mSec. Such shorter deblock time periods help prevent the addition of more than one nucleotide to the primer prior to scanning (e.g., accuracy is enhanced).

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li.sup.+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li.sup.+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca.sup.2+. In addition to Li.sup.+, the fluid can contain polyethylenimine (PEI) with or without betaine.

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li.sup.+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li.sup.+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca.sup.2+. In addition to Li.sup.+, the fluid can contain polyethylenimine (PEI) with or without betaine.

A method including steps of (a) providing an array of sites, wherein each site comprises a mixture of different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment that is selective for a first of the different reversible blocking moieties and a second treatment that is selective for a second of the different reversible blocking moieties.