Genomic DNA was extracted from peripheral blood samples using Blood Genomic Extraction Kit (Qiagen). A panel was designed to cover 261 genes, including 67 known genes responsible for dominant and recessive HSP forms and other 194 genes associated with spastic paraplegia (Table S1). Deep sequencing was performed using Illumina Hiseq2000 system (GrandOmics Biosciences Co). The annotation and analysis of sequenced reads were performed as described previously (Liu et al., 2019 ). Sanger sequencing was used to validate the candidate variants after data analysis. Co‐segregation analysis was conducted through screening for the confirmed variants in the family members. To further screen for large deletions or duplications of culprit genes (SPAST, ATL1, REEP1, PGN, and SPG11), the Multiplex ligation‐dependent probe amplification assay (MLPA) analysis was performed as we described previously (Lu et al., 2018 ). Moreover, analysis of trinucleotide repeats in SCA 1, 2, 3, 6, 7, 12, and 17, as well as Dentatorubral–pallidoluysian atrophy (DRPLA), which could not be detected by targeted sequencing analysis, was conducted via repeat‐primed PCR combined with fragment length analysis.
Blood genomic extraction kit
Manufactured by Qiagen
Sourced in Germany
The Blood Genomic Extraction Kit is a laboratory equipment product designed to extract genomic DNA from whole blood samples. It provides a reliable and efficient method for isolating high-quality DNA for downstream applications.
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9 protocols using blood genomic extraction kit
1
Genetic Screening for Hereditary Spastic Paraplegia
2
Genomic DNA Extraction and ATP7B Sequencing
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Genomic DNA was extracted from peripheral EDTA-treated blood by Blood Genomic Extraction Kit (Qiagen, Hilden, Germany). The Sanger sequencing of ATP7B was performed with a procedure described previously [8 (link)].
3
Genetic Analysis of CYP27A1 Gene
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Genomic DNA was extracted from peripheral blood samples using a commercial blood genomic extraction kit (Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) was carried out to amplify all exons and flanking regions of CYP27A1. Direct Sanger sequencing was performed on an ABI 3500xl Dx Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) as described previously [28 (link)]. The primers for CYP27A1 were listed in Additional file 1 : Table S1. The 1000 Genomes Project (https://www.ncbi.nlm.nih . gov/variation/tools/1000 genomes/) and the ExAC database (https://exac.broadinstitute.org/) were used to check the frequency of variants in the general population. Three software programs, including SIFT (http://sift.jcvi.org/ ), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/ ) and Mutation Taster (http://www.mutationtaster.org/ ) were used to predict the possible protein functional changes caused by the variants.
4
Congenital Nonprogressive Cerebellar Ataxia in Chinese Family
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A non-consanguineous Chinese family including four affected persons (proband, father, sister, and niece), mother (unaffected), and two aunts (unaffected) was enrolled in this study (Fig. 1 a). Among four patients, dominant inheritance history of congenital nonprogressive cerebellar ataxia (CNPCA) associated with delayed motor milestone and cognitive impairment was observed. Genomic DNA was extracted from peripheral EDTA-treated blood using blood genomic extraction kit (Qiagen, Germany) and quantified using a Nano Drop 2000 unit (Thermo Fisher Scientific, Wilmington, DE). This study was approved by the Ethics Committee of China-Japan Friendship Hospital. The methods in this study were performed in accordance with the approved guidelines. Written informed consent was obtained from all the subjects. All the patients underwent a standard neurologic examination conducted by two qualified neurologists.![]()
Pedigree charts of the family and brain magnetic resonance imaging (MRI) of III:2.
5
DNA Extraction and Genetic Analysis
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Extracting DNA from peripheral EDTA‐treated blood was performed using Blood Genomic Extraction Kit (Qiagen) according to the manufactures’ instructions. Sanger sequencing was performed on an ABI 3500xL Dx Genetic Analyzer (Applied Biosystems), and the procedure was described previously.7 , 8 For patients who had only one variant detected by Sanger sequencing, we further performed multiplex ligation‐dependent probe amplification assay (MLPA) using the ATP7B MLPA kit (SALSA P098‐D1, MRC‐Holland).
6
Targeted NGS for Variant Discovery
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Genomic DNA was extracted from peripheral blood samples using the blood genomic extraction kit (Qiagen, Germany) and subjected to targeted next-generation sequencing. The coding exons and flanking sequences of over 20,000 genes were captured for paired-end sequencing (2× 100 bp) on Illumina HiSeq2500 (Illumina, USA). The average sequencing depth of the target region was higher than 170×, with over 95% of bases covered by at least 30×. An in-house genome analysis pipeline composed of BWA 0.7.12 (Li and Durbin, 2009 (link)), GATK 3.5 (McKenna et al., 2010 (link); DePristo et al., 2011 (link)), snpEff 4.0 (Cingolani et al., 2012 (link)), BED Tools 2.25.0 (Quinlan, 2014 ), ANNOVA (Yang and Wang, 2015 (link)) were used to process demultiplexed fastq data. BAM files were generated to visualize read pairs and variant calling in IGV (Broad Institute, USA). Candidate variants were filtered based on the population frequency of 2%, predicated variant pathogenicity, inheritance mode, and protein interaction network.
7
Genetic Screening for Wilson's Disease
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Genomic DNA was extracted using Blood Genomic Extraction Kit (Qiagen, Hilden, Germany) from peripheral EDTA-treated blood. The ATP7B variants of patients and their relatives were verified through Sanger sequencing, with a procedure described in our previous report [24 (link)]. For patients who were detected with only one heterozygous pathogenic variant, we performed multiplex ligation-dependent probe amplification assay (MLPA) with the ATP7B MLPA kit (SALSA P098-D1, MRC-Holland, the Netherlands) [25 (link)].
8
SCA3 Genotyping by PCR and Sequencing
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Genomic DNA was extracted from venous blood samples from participants using Blood Genomic Extraction Kit (Qiagen). Genetic testing was conducted as previously reported (Kawaguchi et al., 1994 ). The CAG repeats number of heterozygous SCA3 was determined by polymerase chain reaction amplification combined with Sanger sequencing as described in our previous studies (Gan et al., 2010 , 2015 ). The CAG repeats number of homozygous SCA3 was conducted using DNA fragment analysis by capillary electrophoresis on an ABI 3730XL DNA analyzer (XiangYin Biotechnology).
9
Comprehensive Genetic Screening for HSP
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Genomic DNA was extracted from peripheral blood samples using Blood Genomic Extraction Kit (Qiagen, Germany). A panel was designed to cover 261 genes, including 67 known genes responsible for dominant and recessive HSP forms and other 194 genes associated with spastic paraplegia (Table S1). Deep sequencing was performed using Illumina Hiseq2000 system (GrandOmics Biosciences Co, China). The annotation and analysis of sequenced reads were performed as we described previously(6). Sanger sequencing was used to validate the candidate variants after data analysis. Co-segregation analysis was conducted through screening for the con rmed variants in the family members. In order to further screen for large deletions or duplications of culprit genes (SPAST, ATL1, REEP1, PGN, and SPG11), the Multiplex ligation-dependent probe ampli cation assay (MLPA) analysis were performed as we described previously (4) . Moreover, analysis of trinucleotide repeats in SCA 1, 2, 3, 6, 7, 12 and 17, as well as Dentatorubral-pallidoluysian atrophy (DRPLA), which could not be detected by targeted sequencing analysis, was conducted via repeat-primed PCR combined with fragment length analysis.
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