Genomic DNA was isolated from fresh-frozen collected tissues (tail, striatum and liver) using DNeasy Blood & Tissue kit (Qiagen, Valencia, CA). Genotyping of the HdhQ111 knock-in allele was carried out using a previously established polymerase chain reaction (PCR) amplification assay, with fluorescently labelled primers (Wheeler et al. 2003 (link)). The size of the PCR products was then determined using the ABI PRISM 3730xl automated DNA Sequencer (Applied Biosystems, Foster City, CA) and GeneMapper version 3.7 software. All runs included the same control DNAs of known Htt CAG repeat size. Somatic instability was quantified from GeneMapper traces as described previously (Lee et al. 2010 (link)). Briefly, the main allele was identified as the highest peak in each analysis and peaks with height less than 20 % of the main allele were excluded. The peak height of each peak was divided by the sum of the heights of all signal peaks and then multiplied by the CAG change relative to the main allele. These values were summed to generate an instability index. To qualitatively assess the different patterns of repeat instability in liver and striatum, the distance (in CAG repeats) between the two modes and the distance (in CAG repeats) between the constitutive repeat mode and the longest repeat was calculated as previously described (Lee et al. 2011 (link)).
Abi prism 3730xl automated dna sequencer
Manufactured by Thermo Fisher Scientific
The ABI PRISM 3730xl is an automated DNA sequencer. It performs high-throughput DNA sequencing analysis. The instrument utilizes capillary electrophoresis technology to analyze DNA samples.
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Lab products found in correlation
Dneasy blood tissue kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Taqman genotyping probes, by Thermo Fisher Scientific (1 mentions)
Genescan 500 rox, by Thermo Fisher Scientific (1 mentions)
Genescan 3, by Thermo Fisher Scientific (1 mentions)
Dye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using abi prism 3730xl automated dna sequencer
1
Quantifying Somatic Instability in Huntington's Disease
2
Genotyping of Huntington's Disease Markers
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Selection of the majority of the markers used in this study was based upon previous work from our lab that showed that they were overrepresented on HD chromosomes. [Lee et al., 2012 ] Genotyping of nine SNPs (rs6814736, rs7667745, rs1751848, rs762847, rs7658462, rs3129317, rs1730768, rs12641989, and rs16844364 ‐ Fig. 1 ) flanking the CAG repeat was performed by real‐time polymerase chain reaction (PCR), using the commercially available Taqman Genotyping probes (Applied Biosystems, Foster City, CA), carried out on the LightCycler® 480 (Roche Diagnostics, Mannheim, Germany), following manufacturer's instructions. Repeat sizes of the HTT CAG repeat, CCG repeat and D4S127 and genotyping of the delta2642 polymorphism were determined using previously established, but slightly modified PCR amplification assays with fluorescently labelled primers. [Taylor et al., 1992 ; Rubinsztein et al., 1993 ; Warner et al., 1993 ; Ambrose et al., 1994 ] The fragments' size was then determined using the ABI PRISM 3730xl automated DNA Sequencer (Applied Biosystems, Foster City, CA) and GeneMapper version 3.7 software. For each marker, a set of sequenced samples was used as standards.
3
Genotyping with Fluorescent Microsatellites
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Ten microsatellite loci located on 10 chromosomes (R158, R8, R29, R203, R102, R7, R137, R36, R60, R145) were selected based on previous information (Hilbert et al., 1991 (link); Serikawa et al., 1992 (link)). For each primer set, the forward primer was 5′‐fluorescent labeled with one of two dyes (6‐FAM or HEX). Amplifications were carried out in 10‐μl reaction volumes, using reagents, concentrations, and procedures for PCR reactions as described (Jing et al., 2014 (link)). Each round included one negative control, and the negative control yielded no product. Product sizes were estimated relative to an internal standard (GeneScan‐500 ROX) in polyacrylamide gels using an ABI PRISM 3730XL automated DNA sequencer, GENESCAN 3.1, and GENOTYPER 2.5 software (all from PerkinElmer Applied Biosystems).
4
Yeast Two-Hybrid Screening of CSFV and BVDV E2 Proteins
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The GAL4-based yeast two-hybrid system was used for this study [21] (link), [22] (link). The ‘bait’ protein, CSFV Brescia E2 protein (amino acid residues 1–342) or BVDV NADL E2 protein (amino acid residues 1–342), was expressed with an N-terminus fusion to the GAL4 Binding Domain (BD). As ‘prey’, the previously described swine macrophage cDNA library and bovine cDNA library containing proteins fused to the GAL4 Activation Domain (AD) was used. Screening was done as previously described [19] (link). Sequencing of the identified library clones was performed by the dideoxynucleotide chain-termination method [23] (link). Sequencing reactions were prepared with the Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Reaction products were sequenced using an ABI PRISM 3730xl automated DNA sequencer (Applied Biosystems, Foster City, CA). The identified sequence was checked to be in-frame with the GAL4-AD and the NCBI BLAST algorithm was used to identify the host protein.
5
Genotyping of Polyglutamine Disorders
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We determined the sizes of CAG repeats of ATN1 [DRPLA MIM #125370], ATXN1 [spinocerebellar ataxia type 1; SCA1 MIM #164400], ATXN2 [spinocerebellar ataxia type 2; SCA2 MIM #183090], ATXN3 [Machado-Joseph disease aka spinocerebellar ataxia type 3; SCA3 MIM #109150], CACNA1A [spinocerebellar ataxia type 6; SCA6 MIM #183086] and TBP [spinocerebellar ataxia type 17; SCA17 MIM #607136] in the participants of the COHORT study (n = 606). After quality control, 551, 502, 604, 503, 497 and 483 COHORT samples were analyzed for ATN1, ATXN1, ATXN2, ATXN3, CACNA1A and TBP, respectively. In addition, REGISTRY samples were analyzed to determine the CAG repeat sizes of ATXN3 (n = 885). CAG repeat lengths for each polyglutamine disease-causing genes were determined by polymerase chain reaction (PCR) assays, using fluorescently labelled primers with minor modifications.48 (link) PCR products were resolved by an ABI PRISM 3730XL automated DNA Sequencer (Applied Biosystems) and analyzed using GeneMapper version 3.7 software. A set of genomic DNA standard samples were also sequenced for each polyglutamine disease-causing repeat and used as references of CAG allele sizes. Expansions above the non-disease associated repeat range were sequenced after gel separation to further confirm the number of CAGs and the presence of CAA/CAT interruptions.
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