We targeted chr4.hg19:46918163-47005187 encompassing 74kb of GABRA4 (NM_000809) and 10kb upstream and 5kb downstream of the gene with long-range PCR. Ten tiled primer pairs each amplifying ~8.7kb were designed using Primer3 software (http://frodo.wi.mit.edu ) [32 (link), 33 (link)] (Online Resource 1) and synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA). PCR was performed using the Expand Long Range dNTPack PCR kit (Roche Applied Science, Indianapolis, IN, USA) on Veriti 96-Well Fast Thermal Cyclers (Life Technologies, Carlsbad, CA, USA). PCR products were visualized on 0.75% agarose-ethidium bromide gel and primer dimer and nonspecific products removed with the NucleoFast 96 PCR kit (Machery-Nagel, Bethlehem, PA, USA) following standard manufacturer protocol for a vacuum manifold.
Protocol detail
Targeted GABRA4 Gene Sequencing
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Corresponding Organization : University of Miami
Other organizations : Case Western Reserve University, Dr. John T. Macdonald Foundation
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Variable analysis
independent variables
- Primer pair design using Primer3 software
- Long-range PCR amplification of targeted region
- Amplified PCR products
- Presence/absence of primer dimers and nonspecific products
- Targeted region on chr4.hg19:46918163-47005187 encompassing GABRA4 gene and 10kb upstream and 5kb downstream
- Expand Long Range dNTPack PCR kit
- Veriti 96-Well Fast Thermal Cyclers
- NucleoFast 96 PCR kit for purification
- None specified
- None specified
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