Emulsion PCR Amplification of DNA Libraries with Degenerate Central Regions for Aptamer Selection

Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

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Evaluation of the diversity of random DNA-libraries by the shape of amplification curves for estimation of the efficiency of aptamer selection

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20196506477 ◽

2019 ◽

Vol 65 (6) ◽

pp. 477-484 ◽

Cited By ~ 1

Author(s):

S.P. Radko ◽

S.A. Lapa ◽

A.V. Chudinov ◽

S.A. Khmeleva ◽

M.M. Mannanova ◽

...

Keyword(s):

Chain Reaction ◽

Dna Library ◽

Aptamer Selection ◽

Smad4 Protein ◽

Dna Libraries ◽

Polymerase Chain ◽

Smad Binding Element ◽

A Site ◽

Oligonucleotide Sequence ◽

Selection Of

Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.

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By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

PLoS ONE ◽

10.1371/journal.pone.0114693 ◽

2014 ◽

Vol 9 (12) ◽

pp. e114693 ◽

Cited By ~ 36

Author(s):

Fabian Tolle ◽

Julian Wilke ◽

Jesper Wengel ◽

Günter Mayer

Keyword(s):

Pcr Amplification ◽

Product Formation ◽

Dna Libraries

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Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation

Molecular Biology ◽

10.1134/s0026893318060110 ◽

2018 ◽

Vol 52 (6) ◽

pp. 854-864 ◽

Cited By ~ 5

Author(s):

S. A. Lapa ◽

K. S. Romashova ◽

M. A. Spitsyn ◽

V. E. Shershov ◽

V. E. Kuznetsova ◽

...

Keyword(s):

Strand Separation ◽

Dna Libraries ◽

Emulsion Pcr

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Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries

Genomics ◽

10.1016/0888-7543(92)90037-s ◽

1992 ◽

Vol 13 (4) ◽

pp. 1192-1197 ◽

Cited By ~ 34

Author(s):

Gal Ehrlich ◽

Evani Viegas-Pequignot ◽

Dalia Ginzberg ◽

Lilian Sindel ◽

Hermona Soreq ◽

...

Keyword(s):

In Situ Hybridization ◽

Somatic Hybrid ◽

Fluorescent In Situ Hybridization ◽

Hybrid Cell ◽

Pcr Amplification ◽

Dna Libraries ◽

Somatic Hybrid Cell ◽

Acetylcholinesterase Gene

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Liquid drop of DNA libraries reveals total genome information

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2017138117 ◽

2020 ◽

Vol 117 (44) ◽

pp. 27300-27306 ◽

Cited By ~ 1

Author(s):

Stanislav S. Terekhov ◽

Igor E. Eliseev ◽

Leyla A. Ovchinnikova ◽

Marsel R. Kabilov ◽

Andrey D. Prjibelski ◽

...

Keyword(s):

Single Molecule ◽

High Throughput Sequencing ◽

Dna Templates ◽

Single Dna Molecules ◽

Emulsion Droplets ◽

Dna Libraries ◽

Emulsion Pcr ◽

Robust Techniques ◽

Genome Information

Conventional “bulk” PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.

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APPLICATION OF GRAPHENE OXIDE ON APTAMER-BASED BIOSENSOR DEVELOPMENT FOR AUTHENTICATION OF GELATIN

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2019v11i2.31122 ◽

2019 ◽

pp. 254-258

Author(s):

HARI WIDADA ◽

ABDUL ROHMAN ◽

RIRIS ISTIGHFARI JENIE ◽

SISMINDARI .

Keyword(s):

Graphene Oxide ◽

Gel Electrophoresis ◽

Dna Analysis ◽

Selection Process ◽

Polyacrylamide Gel Electrophoresis ◽

Pcr Amplification ◽

Native Page ◽

Sharp Separation ◽

Aptamer Selection ◽

Native Polyacrylamide Gel Electrophoresis

Objective: The objective of this study was to perform aptamer selection using systematic evolution of ligands by exponential enrichment (SELEX) method which assisted by graphene oxide against target of porcine gelatin (non-halal gelatin). Methods: The aptamer selection was carried out using SELEX method without target immobilization. Selection of aptamer capable of binding porcine gelatin by applying grafen oxide (GO) was known as GO-SELEX. The selection process was initially carried out by incubation of single-stranded DNA (ssDNA) libraries targeting on porcine gelatin with the addition of graphene oxide. The selected ssDNA was then purified by several stages namely; symmetric PCR amplification, purification of products with DNA purification kits, asymmetric PCR amplification, and continued purification of DNA with native PAGE. The analysis of each stage was done by agarose gel electrophoresis. Results: the results showed that aptamer targeting porcine DNA could be selected. This was indicated by the results of DNA analysis using native polyacrylamide gel electrophoresis (PAGE) in which sharp separation band with a base length equivalent to the marker of the ssDNA library (about 80 base pair) was obtained. Conclusion: Aptamer targeting on porcine gelatin has been successfully developed using GO-SELEX method. GO can increase selectivity in developing aptamer which will be used as a biosensor to detect porcine gelatin. The method could be proposed as a standard of apatamer based method for porcine gelatin detection on halal products authentication.

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