Method and Apparatus for Rapid Nucleic Acid Sequencing

a nucleic acid and sequencing method technology, applied in the field of methods and apparatus for rapid nucleic acid sequencing, can solve the problems of inability to predict the operation of the transmission gate, or to exclude the operation entirely, and the length of individual templates that can be sequenced, and achieve the effects of increasing the number of template nucleic acids used per sensor, and optionally per reaction chamber, and increasing the magnitude of charge chang

Inactive Publication Date: 2011-11-17
LIFE TECH CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0058]In still other methods, the number of template nucleic acids used per sensor, and optionally per reaction chamber, is increased. Since the sequencing-by-synthesis reactions contemplated by the invention typically occur simultaneously on a plurality of identical template nucleic acids, increasing the number of templates increases the number of sequencing byproduct (such as hydrogen ions) released per simultaneous nucleotide incorporation, thereby increasing signal that can be detected. Similarly, increasing the number of templates immobilized to an ISFET surface, as contemplated by some

Problems solved by technology

In particular, because the body and source of the p-channel ISFET are electrically coupled together, implementing the p-channel MOSFET S11P in the same n-well as the p-channel ISFET 50 would lead to unpredictable operation of the transmission gate, or preclude operation entirely.
Analysis of entire genomes of viruses, bacteria, fu

Method used

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  • Method and Apparatus for Rapid Nucleic Acid Sequencing
  • Method and Apparatus for Rapid Nucleic Acid Sequencing
  • Method and Apparatus for Rapid Nucleic Acid Sequencing

Examples

Experimental program
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Effect test

example 1

Bead Preparation

[0718]Binding of Single-Stranded Oligonucleotides to Streptavidin-Coated Magnetic Beads. Single-stranded DNA oligonucleotide templates with a 5′ Dual Biotin tag (HPLC purified), and a 20-base universal primer were ordered from IDT (Integrated DNA Technologies, Coralville, Ind.). Templates were 60 bases in length, and were designed to include 20 bases at the 3′ end that were complementary to the 20-base primer (Table 4, italics). The lyophilized and biotinylated templates and primer were re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) as 40 μm stock solutions and as a 400 μM stock solution, respectively, and stored at −20° C. until use.

[0719]For each template, 60 μl of magnetic 5.91 μm (Bangs Laboratories, Inc. Fishers, Ind.) streptavidin-coated beads, stored as an aqueous, buffered suspension (8.57×104 beads / p L), at 4° C., were prepared by washing with 120 μl bead wash buffer three times and then incubating with templates 1, 2, 3 and 4 (T1, T2, T3, T4: T...

example 2

On-Chip Polymerase Extension Detected by pH Shift on an ISFET Array

[0734]Streptavidin-coated 2.8 micron beads carrying biotinylated synthetic template to which sequencing primers and T4 DNA polymerase are bound were subjected to three sequential flows of each of the four nucleotides. The template sequence downstream of the sequencing primer was a G(C)10(A)10 (SEQ ID NO:5). Each nucleotide cycle consisted of flows of dATP, dCTP, dGTP and dTTP, each interspersed with a wash flow of buffer only. Flows from the first cycle are shown in blue, flows from the second cycle in red, and the third cycle in yellow. As shown in FIG. 72A, signal generated for both of the two dATP flows were very similar. FIG. 72B shows that the first (blue) trace of dCTP is higher than the dCTP flows from subsequent cycles, corresponding to the flow in which the polymerase should incorporate a single nucleotide per template molecule. FIG. 72C shows that the first (blue) trace of dGTP is approximately 6 counts hig...

example 3

Sequencing in a Closed System and Data Manipulation

[0735]Sequence has been obtained from a 23-mer synthetic oligonucleotide and a 25-mer PCR product oligonucleotide. The oligonucleotides were attached to beads which were then loaded into individual wells on a chip having 1.55 million sensors in a 1348×1152 array having a 5.1 micron pitch (38400 sensors per mm2). About 1 million copies of the synthetic oligonucleotide were loaded per bead, and about 300000 to 600000 copies of the PCR product were loaded per bead. A cycle of 4 nucleotides through and over the array was 2 minutes long. Nucleotides were used at a concentration of 50 micromolar each. Polymerase was the only enzyme used in the process. Data were collected at 32 frames per second.

[0736]FIG. 73A depicts the raw data measured directly from an ISFET for the synthetic oligonucleotide (SE ID NO:6). One millivolt is equivalent to 68 counts. The data are sampled at each sensor on the chip (1550200 sensors on a 314 chip) many time...

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Abstract

Methods and apparatus relating to FET arrays including large FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions.

Description

RELATED APPLICATION[0001]This application claims priority under 35 U.S.C. §119(e) to U.S. Provisonal Applications 61 / 196,953, 61 / 198,222, 61 / 205,626, filed Oct. 22, 2008, Nov. 4, 2008 and Jan. 22, 2009, respectively, the entire contents of all of which are incorporated by reference.FIELD OF THE DISCLOSURE[0002]The present disclosure is directed generally to inventive methods and apparatus relating to detection and measurement of one or more analytes including analytes associated with or resulting from a nucleic acid synthesis reaction.BACKGROUND[0003]Electronic devices and components have found numerous applications in chemistry and biology (more generally, “life sciences”), especially for detection and measurement of various chemical and biological reactions and identification, detection and measurement of various compounds. One such electronic device is referred to as an ion-sensitive field effect transistor, often denoted in the relevant literature as ISFET (or pHFET). ISFETs con...

Claims

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Application Information

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IPC IPC(8): C40B20/00
CPCC12Q1/6874G01N27/4145G01N27/4148H01L21/82C12Q1/6869C12Q2565/607
Inventor ROTHBERG, JONATHAN M.HINZ, WOLFGANGDAVIDSON, JOHN F.VAN OIJEN, ANTOINE M.LEAMON, JOHN H.HUBER, MARTIN
Owner LIFE TECH CORP
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