pages tagged PCR

Zhu Y, Zhang YX, Liu WW, Ma Y, Fang Q, Yao B.

Sci Rep. 2015 Apr 1;5:9551. doi:10.1038/srep09551

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot

For the amplification of a miRNA, the authors reported a 98.03% efficiency in standard conditions. It dropped to 90.94% with 0.5 mM 49.99% PBS, 86.96% with 2 mM, 80.47% with 10 mM (1×), and 49.99% with 50 mM. The PCR was done in TaqMan universal PCR master mix.

Bayega A, Oikonomopoulos S, Wang YC, Ragoussis J.

Front Genet. 2022 Oct 17;13:1031355. doi:10.3389/fgene.2022.1031355.

Improved Nanopore full-length cDNA sequencing by PCR-suppression.

“We observed [a range of 2.2 to 8.3-fold increase in yield] of amplicons from the Panhandle method compared to the ONT method.” “Further, the cDNA profile of samples from Panhandle protocol showed a significantly reduced amount of molecules below 600 bp compared to ONT protocol.” “Reads generated with the ONT protocol showed a marked 3′ bias with only about 40–50% of reads showing full-length coverage of the genes”

for each sample total RNA was added together with

RNA + 1 μL of 10 μm oligo (dT) primer + 1 μL of 10 mm dNTPs + Final volume: 11.6 μL pre-RT reaction.

the reaction was incubated at 72°C 3 min. 4°C 10 min, 25°C 1 min, then held at 4°C.

A 10.4μL reverse transcription (RT) reaction containing 1 X Maxima H Buffer, 1 μL RNaseOut (NEB), 2 μL of 100 μm TSO, 2 μL of 5M Betaine (Sigma-Aldrich), and 1 μL of Maxima H reverse transcriptase was added to the pre-RT reaction and the reaction incubated as shown in Supplementary Protocol.

Following reverse transcription,

5 μL of cDNA was used in a 50 μL PCR reaction containing 1 μL of 10 μm PCR primer and 25 μL of 2x LongAmp Taq Master mix (NEB).

PCR was performed as shown in Supplementary Protocol. 20 PCR cycles were used. Following PCR, 1 μL of exonuclease (NEB) was added to each reaction and incubated for 15 min at 37°C followed by 15 min at 80°C and the samples were purified using 1x AMPure XP beads (Beckman Coulter).

Terekhov SS, Eliseev IE, Ovchinnikova LA, Kabilov MR, Prjibelski AD, Tupikin AE, Smirnov IV, Belogurov AA Jr, Severinov KV, Lomakin YA, Altman S, Gabibov AG.

Proc Natl Acad Sci U S A. 2020 Oct 21:202017138. doi:10.1073/pnas.2017138117.

Liquid drop of DNA libraries reveals total genome information.

“[...] either 3% of Abil EM 180 (A-oil) or a mixture of 4.5% Span 80/0.4% Tween 80/0.05% Triton X-100 (T-oil) was used [...] The homogeneity of the amplified DNA was much better in the case of A-oil, whereas ePCR in T-oil resulted in the formation of high-molecular-weight by-products [...] Thirty-five cycles of ePCR in A-oil were sufficient to reach saturation in the majority of the droplets. T-oil was less stable, displaying droplet coalescence after 25 cycles [...].” “Appearance of clear amplicon bands on electropherograms does not correlate with the uniformity of amplification during ePCR.” “ePCR clearly outperformed bulk PCR, reducing the number of reads with gross errors by twofold [and] resulted in a more uniform distribution of amplified sequences”

Salk JJ, Sanchez JA, Pierce KE, Rice JE, Soares KC, Wangh LJ.

Anal Biochem. 2006 Jun 1;353(1):124-32 doi:10.1016/j.ab.2006.02.012

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR.

The PPi produced during the PCR can be removed with the pyrosequencing enzymes themselves. The antarctic phosphatase from NEB was not very efficient at this task.

Nucleic Acids Res. 2001 May 1;29(9):e45.

Pfaffl MW

A new mathematical model for relative quantification in real-time RT-PCR.

Method of normalisation taking PCR efficiency into account.

Nucleic Acids Res. 1999 Sep 15;27(18):e23.

Shagin DA, Lukyanov KA, Vagner LL, Matz MV.

Regulation of average length of complex PCR product.

Suppression PCR amplification of a phage lambda digested by HindII on which inverted terminal repeats were ligated. Suppression effect is stronger when the PCR primer is shorter than the terminal repeats, and when the PCR primer molarity is lower (tested in a 750–0 nM range).

Nakano M, Komatsu J, Matsuura S, Takashima K, Katsura S, Mizuno A.

J Biotechnol. 2003 Apr 24;102(2):117-24.

Single-molecule PCR using water-in-oil emulsion.

Uses the Triton X-100 contained in the PCR buffer as a surfactant.

Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):596-601 doi:10.1073/pnas.012372799

Hogrefe HH, Hansen CJ, Scott BR, Nielson KB.

Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation.

dTTP is deaminated to dUTP during PCR, which causes the synthesis of uracil-containing DNA, a potent inhibitor of archeal DNA polymerases.

Sun Z, Chen Z, Hou X, Li S, Zhu H, Qian J, Lu D, Liu W.

PLoS One. 2008;3(11):e3701. doi:10.1371/journal.pone.0003701

Locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification.

Genomic DNA amplified in a collection of fragments mimicking restriction digests using LNA pentamers and the Stoffel fragment of the Taq polymerase. Annealing at 40 °C and extension at 50 °C.

H. Christina Fan, Glenn K. Fu, Stephen P. A. Fodor

Science. 2015 Feb 6;347(6222):1258367 doi:10.1126/science.1258367

Combinatorial labeling of single cells for gene expression cytometry.

Multiplex PCR. Microwells in 5% agarose.

Shugay M, Britanova OV, Merzlyak EM, Turchaninova MA, Mamedov IZ, Tuganbaev TR, Bolotin DA, Staroverov DB, Putintseva EV, Plevova K, Linnemann C, Shagin D, Pospisilova S, Lukyanov S, Schumacher TN, Chudakov DM

Nat Methods. 2014 Jun;11(6):653-5. doi: 10.1038/nmeth.2960

Towards error-free profiling of immune repertoires.

Correction of early PCR errors, assuming that they should also be seen frequently as late errors.