Expand high fidelity pcr system kit

Specific primers for amplifying and sub-cloning DvCad1-CR8 (⁹⁶¹V-I¹⁰⁸⁵) and -CR10 (¹²¹⁹Q-W¹³²⁹) fragments from pET-30/DvCad1-CR8-10 [14 (link)] are listed in Appendix A. Polymerase chain reaction (PCR) amplifications were performed using the Expand High Fidelity PCR System kit (Roche Diagnostics, Indianapolis, IN) with pET-30/DvCad1-CR8-10 as a template, through 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min. The strategy for deleting a single fragment from DvCad1-CR8-10 was based on the Stratagene Quick Change method, as described in [11 (link)]. Briefly, 5’- and 3’- primer pairs containing terminal EcoRI cleavage sites were designed, complementary to the region to be deleted. The amplicon was gel purified (Qiagen) and digested with DpnI and EcoRI, ligated, and the DNA product transformed into E. coli BL21. The coding regions of all deleted plasmids were sequenced. The DvCad1-CR9 (¹¹⁰¹Q-F¹²¹⁸) fragment was cloned by PCR, using the pET-30/DvCad1-CR8-10 plasmid as a template with CR9/FWD, which had an EcoRI site, and CR9/REV, which had a HindIII site. The PCR product was inserted between the EcoRI and HindIII cleavage sites of plasmid pET-30a (+) (Novagen, Madison, WI) and transformed into the E. coli BL21 after ligation. The coding sequences were confirmed through sequencing (Georgia Genomics Facility, University of Georgia, USA).

Amplification and sequencing protocols were conducted as previously described by Kafando et al.³⁰ Briefly, total nucleic acids were extracted from 100 μL of serum using an automated BioRobot MDx extraction platform. HIV-1 RNA was amplified using the Superscript III One-Step RT-PCR system with Platinum^® Taq DNA polymerase (Invitrogen, Thermo Fisher Scientific, and Carlsbad, CA) with primers env-up forward (5′-GTTTCTTTTAGGCATCTCCTATGGCAGGAAGAAG-3′, HXB2 positions 5957–5983) and env-lo reverse (5′-GTTTCTTCCAGTCCCCCCTTTTCTTTTAAAAAG-3′, HXB2 positions 9063–9088).^{31 (link)} Nested amplification was performed using the Expand™ High Fidelity PCR System Kit (Roche Diagnostics, Indianapolis). Primers E60F forward (5′-TAATCAGTTTATGGGATCAAAGC-3′, HXB2 positions 6547–6569)³² and E55R reverse (5′-GCCCCAGACTGTGAGTTGCAACAGATG-3′, HXB2 positions 7940–7914),^{33 (link)} were used, generating PCR products covering ≈1,400 bp of the env gene. Amplification conditions, library preparation, and Illumina MiSeq next-generation sequencing (NGS) were conducted as previously described.³⁰ Following quality control with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc), raw sequence reads were de novo assembled using the Iterative Virus Assembler (IVA)^{34 (link)} to generate a consensus sequence.

Once the deletions' breakpoints had been narrowed down to a sufficiently small region by qPCR, primer sites in the regions immediately flanking the breakpoints were selected for long‐range PCR amplification. As the precise size of the junction fragment in each patient was unknown, several different PCR conditions were tested and optimized. Long‐range PCR was performed with the Expand High Fidelity PCR System kit (Roche, Mannheim, Germany). This protocol was carried out in accordance with the manufacturer's instructions on a SimpliAmp Thermal cycler (Applied BioSystems, Waltham, MA). The PCR products were sequenced using a Big‐Dye® Terminator version 3.1 Cycle Sequencing Kit in an Applied Biosystems 3,730/DNA Analyzer (Applied BioSystems, Waltham, MA). The raw data were analyzed with Chromas trace viewer (http://technelysium.com.au/wp/chromas/). The sequences of the junction fragments were aligned to the reference sequence of MECP2 (NM_004992.3) using Genomatix diAlign® program (local multiple alignment; http://www.genomatix.de/cgi-bin/dialign/dialign.pl).