Efficient methods and compositions for multiplex target amplification PCR

Inventors

Wang, Chunlin • MA, Zhihai • Gharizadeh, Baback

Assignees

Chapter Diagnostics Inc

Abstract

The present disclosure relates to methods of enzymatic treatment of double-stranded PCR amplified products for eliminating or minimizing primer-dimers in multiplex PCR reactions and for the efficient ligation of adapters. The present disclosure relates to methods and compositions that allow more efficient highly multiplex target amplification compared to conventional methods, compositions and kits by minimizing laboratory steps, eliminating primer-dimers and increasing the efficiency of adapter ligation. The disclosed methods use multiple target-specific primers for specific and selective amplification of targets in a subject's genome. The disclosed methods can be used for numerous downstream procedures and analysis, including DNA sequencing.

Core Innovation

The invention provides a method of enriching nucleic acid target sequences in a sample using methylated universal auxiliary primer designs combined with methylation-dependent endonuclease digestion. In a test reaction, two or more target-specific primers are hybridized to target sequences, where the target-specific primers comprise a methylated universal auxiliary portion with a methylation-dependent endonuclease restriction enzyme recognition site and a target-specific portion configured to target the nucleic acid target sequences in the sample. The test reaction is subjected to amplification to produce an amplified product comprising an amplicon.

The amplified product is then digested with a methylation-dependent endonuclease restriction enzyme to form a digestion product, where the digestion product comprises amplicons comprising sticky ends on each end of the strands. Size selection purification is performed on the digestion product to remove digested primer-dimers and unused primers to produce digested amplicons comprising dsDNA. Universal adapters are ligated to the dsDNA from the digested amplicons to form a ligation product, where the ligating universal adapters comprise a universal sequence portion and sticky ends.

Finally, the ligation product is subjected to amplification with barcoded universal primers complementary to a sequence on the ligating universal adapters to form a final amplification product comprising final amplicons. The second independent approach uses universal auxiliary primers for first amplification, followed by a second amplification using a methylated universal auxiliary primer comprising a restriction enzyme recognition sequence, and then methylation-dependent endonuclease digestion to generate sticky-end amplicons.

Both approaches support multiplex enrichment by producing amplicons with sticky ends, removing primer-dimers by size selection, ligating universal adapters, and using barcoded universal primers for final amplification.

Claims Coverage

The document includes two independent claims. Each independent claim centers on producing sticky-end amplicons via methylation-dependent endonuclease digestion from methylated universal auxiliary primer designs, followed by size selection cleanup, universal adapter ligation, and final amplification using barcoded universal primers; the second independent claim additionally structures the workflow with first and second amplifications using universal auxiliary primers and a methylated universal auxiliary primer.

Across both independent claims, the core inventive structure is consistent: universal-auxiliary primer designs that include a methylation-dependent restriction enzyme recognition context, methylation-dependent endonuclease digestion to generate sticky ends, size selection purification to remove digested primer-dimers and unused primers, ligation of universal adapters with sticky ends and a universal sequence portion, and final amplification using barcoded universal primers. The second independent claim additionally emphasizes a workflow split into first amplification with universal auxiliary primers and second amplification with a methylated universal auxiliary primer, before the shared methylation-dependent digestion and universal adapter ligation steps.

Stated Advantages

Documented Applications