CN-119955908-B - Combined liquid and solid phase DNA amplification

Abstract

The present application relates to combined liquid and solid phase DNA amplification, and in particular describes methods for efficiently amplifying and detecting certain nucleic acid sequences in a population. The selected population may be further characterized, for example, by sequencing. The method includes combined liquid phase and solid phase amplification.

Inventors

• David Alan Davidson
• Graham Worsley
• Jarrett Kilparker
• SAMUEL REID

Assignees

• DNAE诊断有限公司

Dates

Publication Date

20260508

Application Date

20200507

Priority Date

20190508

Claims (10)

1. 1. A method for amplifying and analyzing nucleic acid sequences for non-disease diagnostic purposes, the method comprising: a. Taking a system having a liquid portion, wherein the liquid portion comprises a nucleic acid sample, two or more non-immobilized amplification primers and reagents for nucleic acid amplification, and one or more immobilized amplification primers, wherein the non-immobilized amplification primers are in excess relative to the immobilized amplification primers; b. performing an asymmetric nucleic acid amplification reaction using the non-immobilized amplification primer to produce a first non-immobilized nucleic acid amplification product; c. Further amplifying the first non-immobilized nucleic acid amplification product using the one or more immobilized amplification primers to produce an immobilized second nucleic acid amplification product; d. interrogating in real time a localization signal generated at said immobilized second nucleic acid amplification product, and E. Detecting the presence, absence or sequence of the second nucleic acid amplification product.
2. 2. The method of claim 1, wherein the ratio of the non-immobilized amplification primer to the immobilized amplification primer is 3:1.
3. 3. The method of claim 1, wherein additional primers are released or added during the amplification reaction or at a set point in time.
4. 4. The method of claim 1, wherein the non-immobilized amplification primers in the liquid portion are released from the solid phase prior to amplification.
5. 5. The method of claim 1, wherein the amplification product is optically detected.
6. 6. The method of claim 1, wherein the detection of the amplification product comprises a fluorescent reporter probe and a subsequent fluorescent assay.
7. 7. The method of claim 1, wherein the immobilized amplification primer amplifies an interior segment of the first non-immobilized nucleic acid amplification product such that the immobilized second nucleic acid amplification product is shorter than the first non-immobilized nucleic acid amplification product.
8. 8. The method of claim 1, wherein the liquid portion is exposed to more than two immobilized amplification primers, wherein each primer amplifies a different sequence.
9. 9. The method of claim 8, wherein the two or more immobilized amplification primers differ by a single base, thereby detecting a single base variant in the first non-immobilized nucleic acid amplification product.
10. 10. The method of claim 1, wherein the method is performed on an arrayed optical imaging system comprising a surface in contact with the liquid portion.

Description

Combined liquid and solid phase DNA amplification The present patent application is a divisional application of patent application with application number 2020800342267, application day 2020, and entitled "combined liquid and solid phase DNA amplification". Technical Field The present invention relates to methods for sequence-specific amplification of a subpopulation of nucleic acid fragments from a population of broader nucleic acid sequences. The present invention specifically detects only the desired target sequence. Aspects of the invention relate to nucleic acid constructs for use in such methods. The present invention describes methods for efficient targeted amplification and detection of specific nucleic acid sequences in a population. Background Identification of pathogens in blood and other biological samples is critical for effective treatment of a variety of disease states including sepsis. In the uk, 5 people die per hour from sepsis, and 25% of all sepsis survivors suffer from permanent, life-altering consequences. Early detection and identification of pathogens of bacterial infection is necessary to prevent the onset of sepsis and to successfully treat sepsis. Existing methods for detecting and identifying blood-borne infections include blood culture and the use of antibiotic susceptibility tests. These methods involve the cultivation of cells, which is expensive both in time and money. Typically, septic shock will occur before cell culture results can be obtained. The use of existing molecular detection methods, such as PCR, that can identify pathogens in a sample by analyzing their nucleic acids is limited by a low level of overall sensitivity. This is especially a problem when analyzing human samples, since there are large amounts of non-target (human) nucleic acids. These non-target nucleic acids can inhibit downstream purification and amplification of the target (pathogen) nucleic acid, leading to false negative results, or give false positive readings of pathogen nucleic acid due to non-specific amplification. For example, there are several antibiotic resistance genes that have multiple variants due to point mutations or other polymorphisms, and wherein the presence of a particular mutation may result in a different therapeutic strategy than another mutation. It is therefore important to conduct the identification of these polymorphisms in a timely and cost-effective manner. Existing identification strategies using PCR and other amplification reactions have limited ability and laboratories can employ sequencing to elucidate internal sequences and identify polymorphisms, which are time consuming and expensive to implement. Thus, populations that selectively amplify target nucleic acids relative to non-target nucleic acids are of great value to the pharmaceutical industry and research. The amplification reaction typically relies on a pair of amplification primers for each desired sequence to be amplified. Although several pairs of non-immobilized primers can be combined, combining a large number of primer pairs is often not feasible due to cross hybridization between the primers. Thus, multiplex PCR is typically performed using separate primers in separate solution portions, requiring a large number of samples. The present invention describes improvements to amplification reactions that enable sequence selective amplification of a plurality of different primers in a single sample size. Also disclosed are methods for emulsion PCR, wherein a single template is diluted such that the blisters in the oil emulsion contain fewer than one template molecule per blister on average. May include a bead with a single immobilized primer, the second primer being in solution. Thus, amplification is performed using a mixture of two primers, one of which is immobilized and the other in solution. However, in contrast to the further amplification of the first amplification product in the methods described herein, it only produces a single amplification product in each reaction section. Amplification methods are also known in which both primers are immobilized, such as bridge amplification based on the Illumina cluster. In this case, there is no primer in the free solution. Disclosure of Invention The present invention provides a biphasic amplification reaction, including both solid and liquid phase (thermocycling or isothermal) amplification reactions, which can use a combination of immobilized and non-immobilized primers and target DNA templates to simultaneously generate DNA amplicons in solution and on the surface. The presence or absence of amplicons can be detected in an end-point assay after amplification or in real-time. This biphasic approach differs from the prior art, including emulsion PCR, in that it includes both solid (i.e., surface-based) and liquid phase amplification. The nucleic acid sample may be amplified using one or more non-immobilized primers. The liquid phase generated DNA and target