We validated all de novo variant calls reported in CDK13, CHD4 and PRKD1 using capillary sequencing. Primers were designed to amplify 400-600bp products centered on the site of interest. Primer3 design settings were adjusted as follows: primer length - 18 bp +/–3, GC Clamp=1, Tm 60 +/–2, using a human mispriming library. Genomic DNA from all trio members, amplified by Whole Genome Amplification (WGA) using illustra Genomiphi HY or V2 Amplification Kits (GE Healthcare), was used as template DNA in the site-specific PCR reactions. PCR reactions were carried out using Thermo-Start Taq DNA Polymerase (Thermo Scientific), following the manufacturer’s protocol. The PCR products were assessed by Agarose gel electrophoresis and submitted for sequencing to the Faculty Small Sequencing Projects (WTSI core facility). Capillary sequence traces from all trio members were aligned and viewed using an in-house designed web-based tool and scored for the presence or absence of the variant.
Thermo start taq dna polymerase
Manufactured by Thermo Fisher Scientific
Thermo-Start Taq DNA Polymerase is a thermostable DNA polymerase used for PCR amplification of DNA fragments. It possesses 5'-3' DNA polymerase activity and is inactive at lower temperatures, providing improved specificity and sensitivity for PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Genomiphi hy or v2 amplification kits, by GE Healthcare (2 mentions)
M mlv rt enzyme, by Thermo Fisher Scientific (1 mentions)
Maxima sybr green qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Ceqtm 8000 genetic analysis system, by Beckman Coulter (1 mentions)
Gexp starter kit, by Beckman Coulter (1 mentions)
G storm gs1 thermal cycler, by Beckman Coulter (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Chromas lite, by Technelysium (1 mentions)
Genomelab gexp start kit, by Beckman Coulter (1 mentions)
8 protocols using thermo start taq dna polymerase
1
Validating Variant Calls in Cancer Genes
2
Transcriptional Analysis of Cancer Genes
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1μg of total RNA was subjected to reverse transcription, during 1 hour at 37°C, using 1 μg of nonspecific hexameric random primers dN, 1mM dNTP, 10 mM dithiothreitol, 24 units RNaseOUT and 200 units of M-MLV-RT enzyme (Invitrogen). The PCR amplification was performed on 1:10 (v/v) of the 1:10-diluted reverse transcription reaction using 0.2 mM dNTP, 2.5 mM MgCl2 and 1 unit of Thermostart Taq DNA polymerase (Thermo Scientific), and 25 pmol of each specific primer :
NTS (5'-CAGCTCCTGGAGTCTGTGCT-3' and 5'-GAGTATGTAGGGCCTTCTGGG-3')'
NTSR1 (-5'-CGTGGAGCTGTACAACTTCA-3 and 5'-CAGCCAGCAGACCACAAAGG-3)
HER3 (5'-ATGGGGAACCTTGAGATTGTGCT-3' and 5'-ACAGCTTCTGCCATTGTCCT-3')
EGFR (5'-TTTCGATACCCAGGACCAAGCCACAGCAGC-3' and 5' AATATTCTTGCTGGATGCGTTTCTGTA-3')
HER2 (5'-GTGCTAGACAATGGAGACC-3' and 5'-CACAAAATCGTGTCCTGGTAGC-3')
18S (5'-AGGAATTGACGGAAGGGCAC-3' and 5'-GTGCAGCCCCGGACATCTAAG-3')
36B4 (5'-GTGCAGCCCCGGACATCTAAG-3' and 5'-GATTGGCTACCCAACTGTTG-3')
3
Multiplex GeXP Assay for Somatotropic Markers
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Customised GeXP multiplex assays were designed to genes that included the natriuretic peptide system as well as somatotrope markers (see Supplementary Table S1 ). Target-specific reverse transcription (using 100 ng RNA as template), and PCR amplification were performed as we described previously [15 (link),43 (link),44 (link)], and in accordance with manufacturer’s instructions (Beckman Coulter, High Wycombe, UK). In brief, a master mix was prepared for reverse transcription as detailed in the GeXP starter kit (Beckman Coulter, High Wycombe, UK) and performed using a G-storm GS1 thermal cycler (Agilegene Technologies Ltd., Somerton, Somerset, UK), using the following protocol: 48 °C 1 min; 42 °C 60 min; and 95 °C 5 min. From this an aliquot of each reverse transcriptase reaction was added to PCR master mix, consisting of GenomeLab kit PCR reaction mix and Thermo Scientific Thermo-Start Taq DNA polymerase. PCR reactions were performed using G-storm GS1 thermal cycler with a 95 °C activation step for 10 min, followed by 35 cycles of 94 °C 30 s; 55 °C 30 s; 70 °C 60 s. Products were separated and quantified using CEQTM 8000 Genetic Analysis System, and GenomeLab Fragment Analysis software (Beckman Coulter, High Wycombe, UK).
4
PCR amplification and sequencing
5
SF3B1 Exon 14 Sanger Sequencing
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Exon 14 of SF3B1 was sequenced using PCR-based capillary Sanger sequencing in an additional twenty M3-UM with unusual nBAP1+ protein expression [21 (link)]. Oligonucleotides were constructed by Eurofins Genomics; forward 5’-GGCCGAGAGATCATTTCT-3, reverse 5’-AAGAAGGGCAATAAAGAAGGA-3’, product size 289bp. PCR was performed in a reaction volume of 50 μL containing 100 ng of genomic DNA, 0.25 μL of Thermo-Start Taq DNA Polymerase (Thermo Scientific), 5 μL of HP Buffer, 4 μL of 25 mM MgCl2, 2 μL of dNTP (2 mM each), 31.25 μL Nuclease Free water and 1 μL of each of the primers. The thermal cycling profile was as follows: initial denaturation at 95 °C for 15 min and 35 rounds of amplification at 95 °C for 15 s, 55 °C for 30 s and 72 °C for 1 min. A final extension step at 72 °C for 5 min was added. PCR products were purified using the QIAquick PCR purification kit (Qiagen, United Kingdom) according to the manufacturer’s protocol. Sequencing of PCR products was carried out by GATC at Eurofins Genomics in accordance with ISO 17025. Sequencing data were analysed using Chromas Lite (2.1.1., Technelysium Pty Ltd.).
6
Targeted Amplification of ZNF407 and RNF146
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DNA fragments of ZNF407 and RNF146 were amplified using 30 ng genomic DNA as a template and the primers listed below. Due to its size, we covered the first exon of ZNF407 by four separately amplified regions, named A, B, C and D. ZNF407 (Exon 1 A):
5’-AAAGGGTGTTTCATTGGGGC-3’
5’-CACGCTTCCAGGTGTTTCAGC-3’
5’-CTTCTTGTTGAAATGATGCCTTCC-3’
5’-TGATAGTCTTGACCATGCCGAAG-3’
5’-GGAAAAGCATCTCAGGAAGAACC-3’
5’-TTTGTTGATGCTCTTCTTCAGGC-3’
5’-CTTCAGAGCCAGAGGACTTCG-3’
5’-ATGAGCAGCCACAGGTAGGG-3’
5’-GGTGGTTAGTGTTTTCATAATTG-3’
5’-CACCCCAACTCCCAAAATGC-3’
7
Validating Variant Calls in Cancer Genes
8
Multiplex RT-PCR Gene Expression Analysis
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The transcriptional level of each gene was determined using multiplex RT-PCR. The multiplex PCR was operated using a GenomeLab GeXP start kit (Beckman Coulter) with Thermo-StartTaqDNA polymerase (Thermo Fisher Scientific) according to the protocol provided. The amplified products were separated by capillary electrophoresis based on the GenomeLab GeXP Genetic Analysis System. The data were analyzed and normalized against a reference gene and housekeeping genes using the system software. The results were exported for further analysis in a tab-delimited format file.
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