Blood samples were obtained from all family members. We recruited 100 healthy individuals from the same geographical areas as the patients to clarify whether the possible variant was an innocuous polymorphism or pathogenic variant. Genomic DNA was extracted from the blood samples using a DNA Isolation Kit for Mammalian Blood (Tiangen Biotech, China).
Dna isolation kit for mammalian blood
Manufactured by Tiangen Biotech
Sourced in China
The DNA Isolation Kit for Mammalian Blood is a laboratory product designed to extract and purify DNA from mammalian blood samples. It provides a standardized protocol and the necessary reagents to efficiently isolate high-quality genomic DNA from blood, which can be used for various downstream applications such as PCR, sequencing, and genetic analysis.
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9 protocols using dna isolation kit for mammalian blood
1
Familial Genetic Variant Analysis
2
Genetic Analysis of FBN2 Gene Variants
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Blood samples were obtained with informed consent from the family members and from 100 normal individuals. Genomic DNA was extracted from the blood samples using a DNA Isolation Kit for Mammalian Blood (Tiangen Biotech, China). Primer sets were used to amplify 65 exons (exon 1–65) of the FBN2 gene (NM_001999) for sequencing. The sequences of the forward and reverse primers of exon 32 were 5′ TACTGGAAAGTGGCTGAC 3′ and 5′ CAAGGCATACTGTTGAAAT 3′, respectively. The PCR conditions for the DNA amplification were as follows: denaturing at 95 °C for 5 min; 30 cycles of denaturing at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s; and a final step for 5 min at 72 °C. The variation was further confirmed by digestion with the restriction enzyme MunI. The digested products were separated and analyzed by 8% polyacrylamide gel electrophoresis and silver staining.
3
Genotyping of ZPR1 Region SNPs
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We searched for all SNPs in the ZPR1 region with minor allele frequencies (MAF) ≥0.01 using the dbSNP database (Build 143) of NCBI (Build 38). Using this strategy, 24 SNPs were identified (rs964184, rs11823543, rs17120029, rs11604424, rs1942478, rs139753514, rs12286037, rs4417316, rs12274192, rs6589566, rs7483863, rs2075290, rs618923, rs603446, rs76341142, rs11355367, rs74662600, rs10750096, rs3741298, rs140050044, rs12285095, rs2075294, rs33984246, and rs2266788). These 24 SNPs, which completely covered the region of ZPR1 (Fig. 1 ), were included in further analyses.
DNA was extracted from whole blood according to the standard protocol for the DNA Isolation Kit for Mammalian Blood (Tiangen Biotech Co., Ltd, Beijing, China). DNA was stored at −20 °C for SNP analyses. Genotyping was performed for all SNPs using the MassARRAY platform (Sequenom, San Diego, California, US). Briefly, the SNPs were genotyped using high-throughput, matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) mass spectrometry. Next, the resulting spectra were processed using the MassARRAY Typer Analyzer software (Sequenom, San Diego, California, US), and genotype data were generated from the samples. For quality control, both the case and control status were blind during all genotyping processes. Meanwhile, 5% of random samples were repeated, and the results were 100% concordant.
DNA was extracted from whole blood according to the standard protocol for the DNA Isolation Kit for Mammalian Blood (Tiangen Biotech Co., Ltd, Beijing, China). DNA was stored at −20 °C for SNP analyses. Genotyping was performed for all SNPs using the MassARRAY platform (Sequenom, San Diego, California, US). Briefly, the SNPs were genotyped using high-throughput, matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) mass spectrometry. Next, the resulting spectra were processed using the MassARRAY Typer Analyzer software (Sequenom, San Diego, California, US), and genotype data were generated from the samples. For quality control, both the case and control status were blind during all genotyping processes. Meanwhile, 5% of random samples were repeated, and the results were 100% concordant.
4
Genetic Variant Identification in Inherited Retinal Disease
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Peripheral venous blood (5 ml) of the participating family members was sampled, and genomic DNA was extracted with a DNA isolation kit for mammalian blood (Tiangen, Beijing, China). Venous blood and genomic DNA samples were stored at −80 °C before use. The proband (III:2) underwent next-generation sequencing captured with 532 inherited genes related to the genetic visual system. The exon regions of these genes were particularly enriched with a biotinylated capture probe (Joy Orient Translational Medicine Research, Beijing, China). Burrows-Wheeler Aligner (BWA) software was used to perform short-read mapping and alignment, while SOAPsnp software and GATK Indel Genotype were used to test single nucleotide polymorphism (SNP) and insertion and deletion mutations, respectively. Identified mutants were filtered in databases. Sanger sequencing verified whether any remaining variants were cosegregated with the family disease phenotype. The novel mutations in GUCA1A were also genotyped with Sanger sequencing in the 200 normal control subjects. The possible pathogenicity of the mutations was predicted using the Sorting Intolerant from Tolerant (SIFT ) algorithm, Polymorphism Phenotyping v2 (PolyPhen-2 ), Protein Variation Effect Analyzer (PROVEAN ), and MutationTaster .
5
Linkage Analysis of VHL Gene
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Peripheral blood leukocytes were collected from 33 of the 38 living family members, and human genomic DNA was extracted using the DNA Isolation Kit for Mammalian Blood (Tiangen Biotech Co., Ltd., Beijing, China). We genotyped three polymorphic microsatellite markers that flanked VHL on chromosome 3p25 (D3S3691, D3S1597 and D3S1263). The microsatellite markers were amplified by PCR using fluorescently labeled primers. The products were analyzed with an Applied Biosystems 3730 Genetic Analyzer (Applied Biosystem, Foster City, CA, USA). LOD scores were calculated using the MLINK program of the LINKAGE package 5.1 (Perkin Elmer, Waltham, USA). The parameters used for linkage analysis were autosomal dominant inheritance, complete penetrance, a mutation rate of 0, equal male-female recombination rates, equal allele frequency, and the disease allele frequency of 1 in 10,000.[17 (link)]
6
Exome Sequencing of Five Patients
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Exome sequencing was performed on five patients (II3, II7, III18, III22, and IV9) at Berry Genomics Co. Ltd., Beijing, China. Venous blood (5 ml) was collected from the participants and the total human genomic DNA was isolated with the DNA isolation kit for mammalian blood (Tiangen, Beijing, China). Venous blood and genomic DNA samples were stored at −80 °C before use. The Human All Exon (50 Mb) target enrichment system (Agilent Technologies, Santa Clara, CA, USA) was used for whole exome capture and the HiSeq 2500 Sequencing System (Illumina Inc., San Diego, CA, USA) was used for massive parallel sequencing. The gene sequences for this array are available from the Consensus Coding Sequence Region (CCDS) database (http://www.ncbi.nlm.nih.gov/projects/CCDS/ ).
7
Genomic DNA Extraction from Blood
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Blood samples were obtained from all family members. We recruited 100 healthy individuals from the same geographical areas as the patients to clarify whether the possible mutation was an innocuous polymorphism or pathogenic mutation. Genomic DNA was extracted from the blood samples using a DNA Isolation Kit for Mammalian Blood (Tiangen Biotech, China).
8
DNA Extraction from Blood Samples
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2.1 DNA Extraction: Blood samples were obtained from all family members. We recruited 100 healthy individuals from the same geographical areas as the patients lived, to clarify whether new mutations were innocuous polymorphism or pathogenic mutations. Genomic DNA was extracted from the blood samples using a DNA Isolation Kit for Mammalian Blood (Tiangen Biotech, China).
9
Hereditary Spastic Paraplegia in Han Families
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Four unrelated ethnic Han kindreds (marked as families 1-4) with HSP were included in this study (Figure 1). Clinical information and family histories were collected and neurological examinations were performed by a neurologist according to Harding's criteria (Harding, 1981) . Families 1-3 possessed AD inheritance patterns, and family 4 appeared to segregate an X-linked dominant form of HSP, in which all six living affected individuals were females; four males had died of unknown causes within 10 days after delivery. Written informed consent was obtained from all participants prior to the study. The genetic study, conducted on the members of the four families, was approved by the local Ethics Committee. Human genomic DNA was isolated from whole blood using the DNA Isolation Kit for Mammalian Blood (Tiangen Biotech Co., Ltd., Beijing, China) .
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