Mmlv reverse transcriptase variants

A technology of reverse transcriptase and reverse transcription, applied in the direction of transferase, enzyme, hydrolase, etc., can solve problems such as poor solution

Pending Publication Date: 2019-12-31
10X GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Despite these advances in biological characterization, many challenges remain unaddressed, or relatively poorly addressed, by currently available solutions

Method used

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  • Mmlv reverse transcriptase variants
  • Mmlv reverse transcriptase variants
  • Mmlv reverse transcriptase variants

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0066] Preparation of cell-containing microcapsules can be performed in a number of ways. For example, an air knife droplet or aerosol generator can be used to dispense droplets of precursor fluid into the gelling solution to form microcapsules including single cells or small groups of cells. Likewise, membrane-based encapsulation systems can be used to produce microcapsules comprising encapsulated cells as described herein. Disclosures such as those shown in figure 1 The microfluidic system in can be readily used to encapsulate cells as described herein. In particular, and with reference to figure 1 , an aqueous fluid 112 containing (i) individual cells 114 and (ii) polymer precursor material (not shown) flows into the channel junction 110 where it is degraded by the flow of a non-aqueous fluid 116 Dispensed into droplets 118,120. In the case of an encapsulation method, the non-aqueous fluid 116 may also include an initiator (not shown) to cause polymerization and / or cros...

Embodiment 1

[0313] Example 1. Analysis of cellular RNA using emulsions

[0314] In one instance, as in Figure 9A The procedure shown in , reverse transcription with template switching and cDNA amplification (by PCR) was performed in the case of emulsion droplets. Reaction mix dispensed for reverse transcription and cDNA amplification (by PCR) consisted of 1,000 cells or 10,000 cells or 10 ng RNA, beads with barcoded oligonucleotides / 0.2% Tx-100 / 5x Kapa buffer , 2x Kapa HS HiFi Ready Mix, 4 μM Switching Oligomer and Smartscribe. When cells are present, the mixture is partitioned such that most or all droplets contain a single cell and a single bead. The cells are lysed while the barcoded oligonucleotides are released from the beads, and the poly-dT segment of the barcoded oligonucleotides hybridizes to the poly-A tail of the mRNA released from the cells, as in operation 950 . As in operation 952, the poly-dT segment is extended in a reverse transcription reaction, and as in operation 9...

Embodiment 2

[0316] Example 2. Analysis of cellular RNA using emulsions

[0317] In another example, as in Figure 9A The procedure shown in , reverse transcription with template switching and cDNA amplification (by PCR) was performed in the case of emulsion droplets. The reaction mix dispensed for reverse transcription and cDNA amplification (by PCR) included Jurkat cells, beads carrying barcoded oligonucleotides / 0.2% Triton X-100 / 5x Kapa buffer, 2xKapa HS HiFi Ready Mix , 4 μM switching oligo and Smartscribe. The mixture is partitioned such that most or all droplets contain single cells and single beads. The cells are lysed while the barcoded oligonucleotides are released from the beads, and the poly-dT segment of the barcoded oligonucleotides hybridizes to the poly-A tail of the mRNA released from the cells, as in operation 950 . As in operation 952, the poly-dT segment is extended in a reverse transcription reaction, and as in operation 954, the cDNA transcript is amplified. Therma...

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PUM

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Abstract

Disclosed herein, are compositions, methods, and kits comprising engineered reverse transcription enzymes that exhibit several desired properties such as thermal stability, processive reverse transcription, non-templated base addition, and template switching ability. The engineered reverse transcription enzymes described herein demonstrate unexpectedly higher resistance to cell lysate inhibition,greater ability to capture full-length mRNA transcripts, and demonstrate improved results in small reaction volumes as compared to other engineered reverse transcription enzymes.

Description

[0001] cross reference [0002] This application claims the benefit of US Provisional Application No. 62 / 490,492, filed April 26, 2017, which is hereby incorporated by reference in its entirety. Background technique [0003] Significant advances in the analysis and characterization of biological and biochemical substances and systems have led to unprecedented advances in understanding the mechanisms of life, health, disease and therapy. Among these advances, techniques for targeting and characterizing the genomes that make up biological systems have yielded some of the most groundbreaking results, including advances in the use and development of genetic amplification techniques and nucleic acid sequencing techniques. [0004] Nucleic acid sequencing can be used to obtain information in a wide variety of biomedical situations including diagnostics, prognosis, biotechnology, and forensic biology. Sequencing can involve basic methods, including Maxam-Gilbert sequencing and chain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/22C12N15/33
CPCC12N9/1276C12Y207/07049C07K2319/21C07K2319/50
Inventor 约瑟芬·李塞缪尔·马尔斯杰弗里·麦克德莫特弗朗西斯卡·梅斯基卢斯·蒙特斯克拉劳斯凯瑟琳·法伊弗约瑟夫·弗朗西斯·舒加杰西卡·米歇尔·特里索伦戈·B·西拉尔多
Owner 10X GENOMICS
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