Methods and compositions for isothermal whole genome amplification
Inventors
Zhang, Haichuan • Yao, Zuxu • GUNNING, KERRY
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10041106-B2
Publication Date
2018-08-07
Expiration Date
Abstract
Disclosed are methods and compositions for amplification of genetic material, including isothermal WGA of single cells.
Core Innovation
The invention relates to isothermal whole genome amplification of genomic DNA from a cell, including single-cell genomic amplification. A workflow is described that uses a lysis mixture containing a sample with genomic DNA, exogenous oligonucleotides, and a lysis buffer, followed by incubating the lysis mixture to lyse the cell. The lysed material is then combined with a pH-neutralization solution to prepare a neutralized mixture before amplification under isothermal conditions.
The isothermal whole genome amplification is performed by adding a polymerase, dNTPs, and a salt mixture to the neutralized mixture to prepare an amplification mixture, and then incubating under isothermal conditions to amplify the genomic DNA. The method specifies that red blood cells are present in the sample and that the concentration of red blood cells is about 2,500 to about 50,000 cells per microliter of amplification mixture.
The document also describes approaches directed to improving performance of isothermal whole genome amplification for low-input and single-cell DNA, including reducing high allele dropout and locus dropout, reducing amplification bias and array CGH noise, and reducing template-independent polymerization. Example-enabled components include neutralization with a phosphate salt-based neutralization solution and reduced or omitted heating-denaturation steps, and the use of modified/random primers and modified base pairs to reduce template-independent polymerization.
Claims Coverage
The provided independent claims cover isothermal whole genome amplification workflows that include lysis with exogenous oligonucleotides, pH-neutralization, and isothermal amplification with polymerase, dNTPs, and a salt mixture, while maintaining a defined red blood cell concentration in the amplification mixture. Across the two independent claims, the inventive features focus on (i) using red blood cells within a specified concentration range during isothermal WGA and (ii) preparing many low-genomic-DNA subsamples with less than about one cell average per subsample for amplification under the same constrained red blood cell condition.
Red blood cells in isothermal whole genome amplification with defined concentration
Including a sample containing red blood cells in the lysis mixture for whole genome amplification, and performing isothermal whole genome amplification in which the concentration of red blood cells is about 2,500 to about 50,000 cells per microliter of amplification mixture.
Isothermal whole genome amplification using lysis, pH-neutralization, and polymerase/dNTP/salt mixture
Preparing a lysis mixture of a sample containing genomic DNA and red blood cells, exogenous oligonucleotides, and a lysis buffer; incubating for up to 30 minutes to lyse the cell; preparing a neutralized mixture by adding a pH-neutralization solution following incubation; and performing isothermal whole genome amplification by adding a polymerase, dNTPs, and a salt mixture to the neutralized mixture, followed by incubating the amplification mixture under isothermal conditions.
Aliquoting into many subsamples with less than about one cell average per subsample
Aliquoting at least 300 subsamples of a sample containing cells with genomic DNA and red blood cells, where the subsamples each contain an average of less than about one cell containing genomic DNA and red blood cells, and then processing each subsample through lysis with oligonucleotides and lysis buffer, pH-neutralization with a pH-neutralization solution and an amount of oligonucleotide primers sufficient for amplification, and isothermal whole genome amplification using polymerase, dNTPs, and a salt mixture.
The independent claims emphasize isothermal whole genome amplification workflows that combine lysis with exogenous oligonucleotides, pH-neutralization, and isothermal amplification using polymerase, dNTPs, and a salt mixture, while maintaining red blood cells at about 2,500 to about 50,000 cells per microliter of amplification mixture. One independent claim further centers on aliquoting at least 300 subsamples with an average of less than about one cell containing genomic DNA and red blood cells per subsample.
Stated Advantages
Improves performance of isothermal whole genome amplification for low-input and single-cell DNA versus standard protocols that yield high allele dropout (ADO) and locus dropout (LDO).
Reduces amplification bias and array CGH noise.
Reduces template-independent polymerization.
Improves quality outcomes used for downstream detection by supporting quality control metrics such as ADO, LDO, and fidelity/noise metrics.
Documented Applications
Array comparative genomic hybridization (array CGH) for copy number variations (CNVs) using amplified genomic DNA.
Sequencing for genetic variation using amplified genomic DNA.
Quality control using locus dropout rate, allele dropout rate, and/or fidelity value as evaluated characteristics.
Fetal allele identification from maternal-fetal mixtures, including pre-implantation genetic diagnosis (PGD) context.
Interested in licensing this patent?