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Improvements in or relating to amplification of nucleic acids
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A nucleic acid amplification reaction, nucleic acid technology, applied in the field of amplifying nucleic acid molecules
Active Publication Date: 2020-10-02
LUMIRADX UK LTD
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The results showed that although the "wiggle" reaction started to amplify the target faster than the true isothermal reaction, there was still a delay of about 13 minutes before the fluorescent signal rose above the initial background level
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example 1
[0123] Example 1: Protocol for Testing Temperature Selective Quantitative Amplification Reaction (qSTAR)
[0124] Quantification of gene expression by Selective Temperature Amplification Reaction (STAR) as described in WO 2018 / 002649 or other similarly related DNA / RNA amplification techniques such as PCR, SDA or isothermal amplification techniques will not be reliable . The amount of product produced reached a plateau level not directly related to the amount of target DNA in the initial starting sample. By establishing the zone effect of controlled temperature shuttling on the amplification reaction, quantitative amplification can be achieved using strand-displacing polymerases and nicking endonucleases, where the amplified product is directly related to the initial input amount of DNA, RNA, or other known nucleic acid. relevant. According to the present invention, temperature-selective amplification reactions based on nicking enzymes are referred to herein as temperature-...
example 2
[0140] Example 2: Results using unmodified primers
[0141] To demonstrate the potential of this novel amplification technique, qSTAR was performed using four replicates of each target dilution spanning 6 logs of genomic DNA input, and two replicates for a no-target control (NTC). Experimental results using unmodified primers (i.e., primer molecules without any chemically modified abnormal nucleobases) are shown in Figure 4 middle. The amount of signal for "no target control" (fluorescence minus background) is indicated by a dark line ("ntc"). The amount of signal generated in the presence of 20 cp, 200 cp, 2k, 20k, 200k and 2M copies of the target (Chlamydia trachomatis genomic DNA) is indicated by the corresponding lines.
[0142] The qSTAR reaction shows a linear coefficient of determination for the target input, while also demonstrating improvements in speed, sensitivity, and total fluorescence. Surprisingly and unexpectedly, this improvement and separation between t...
example 3
[0148] Example 3: Results using 2'O-methyl modified primers
[0149] The use of 2'O-methyl modified primers is known to reduce the formation of primer dimers during amplification, as described in US Patent Nos. 6,794,142 and 6,130,038. US 2005-0059003 describes the use of a 2'O-methyl modification located at the 3' end of the SDA primer, showing that Bst DNA polymerase I and derivatives can efficiently utilize 2'-modified ribonucleotides as primers. Nucleotides containing one or more 2' modifications (e.g., 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O -[2(methylamino)-2-oxyethyl], 2'-hydroxyl (RNA), 4'-thio, 4'-CH3-O-2'-bridge, 4'-(CH3)3- Target-specific primer regions of O-2'-bridge, 2'-LNA and 2'-O-(N-methylcarbamate 2'-Suc-OH)) will improve the amplification reaction. Using an enzyme selective temperature shuttle (62°C-57°C) as described in the previous example and 2'-O-methylated single base or string of 2'-O-methylated bases towards the 3' of the primer ...
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Abstract
Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps: (a)mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b)using a nicking enzyme to cause a nick at each of said nicking sites in the strands of the duplex; (c)using a polymerase to extend the nicked strands to as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d)repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that the temperature at which the method is performed is non-isothermal, and subject to shuttling, a plurality of times, between an upper temperature and a lower temperature during the amplification process of steps (b)-(d), wherein at the upper temperature, one of said polymerase or nicking enzyme is more active than the other of said enzymes, such that there is a disparity in the activity of the enzymes, and at the lower temperature the disparity in the activity of the enzymes is reduced or reversed.
Description
technical field [0001] The present invention relates to methods for amplifying nucleic acid molecules, in particular methods for amplifying nucleic acid molecules in a quantitative manner. Background technique [0002] The polymerase chain reaction (PCR) is well known and is a standard technique employed for amplifying nucleic acid molecules. The PCR amplification product is detected at the end of the reaction. The amount of product tended to reach a plateau level at which the amount of product did not increase even if the reaction mixture was left for longer periods of time. Thus, in conventional PCR, the amount of product does not necessarily correlate to the concentration of amplified target sequence initially present in the mixture. [0003] To obtain quantitative data, quantitative PCR ("qPCR") is performed, in which the amount of amplification product produced is monitored or detected in real time (qPCR is therefore also called "real-time PCR" or even "RT-PCR", howev...
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