Rapid Amplification of cDNA Ends for Gene Sequencing

The 5′- and 3′- Rapid Amplification of cDNA Ends (RACE) method was applied to the isolated mRNA (GeneRacer kit; Invitrogen) and reverse transcribed to cDNA using the Oligo(dT)-adapter primer (GeneRacer kit and SuperScript III First-Strand Synthesis SuperMix; Invitrogen). The resultant cDNA was then used as a template to obtain the 5′ and 3′ cDNA ends for nested polymerase chain reaction (PCR) amplification (GeneRacer primers included in the GeneRacer kit) with RHD or TMEM50A cDNA primers (Table S1). No cDNA analysis was done for sample 2, because the cDNA sequence for this RHD deletion had been published before.^{22 (link)}The PCR amplicons were purified and sequenced (BigDye Terminator v3.1; Applied Biosystems, Carlsbad, CA) as described previously.^{32 (link)} Nucleotide sequences were aligned (CodonCode Aligner; CodonCode, Dedham, MA) to NCBI RefSeq NM_016124.4 and nucleotide positions defined using the first nucleotide of the coding sequence of RefSeq NM_016124.4 (RHD isoform 1).

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Srivastava K., Stiles D.A., Wagner F.F, & Flegel W.A. (2017). Two large deletions extending beyond either end of the RHD gene and their red cell phenotypes. Journal of human genetics, 63(1), 27-35.

Publication 2017

GeneRacer kit SuperScript III First-Strand Synthesis SuperMix BigDye Terminator v3.1

Cdna Coding nucleotide sequence Isoform Mrna Nested polymerase chain reaction Nucleotide Nucleotide sequences Oligo Polymerase chain reaction Primers Sequence deletion Synthesis

Corresponding Organization :

Other organizations : National Institutes of Health Clinical Center, German Red Cross

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Variable analysis

independent variables

Application of the 5′- and 3′- Rapid Amplification of cDNA Ends (RACE) method to the isolated mRNA

dependent variables

Obtaining the 5′ and 3′ cDNA ends for nested polymerase chain reaction (PCR) amplification

control variables

Reverse transcription of mRNA to cDNA using the Oligo(dT)-adapter primer
Use of the GeneRacer kit and SuperScript III First-Strand Synthesis SuperMix for reverse transcription
Use of the GeneRacer primers for nested PCR amplification
Use of RHD or TMEM50A cDNA primers for nested PCR amplification
Purification and sequencing of the PCR amplicons using BigDye Terminator v3.1 and Applied Biosystems
Alignment of nucleotide sequences to NCBI RefSeq NM_016124.4 and definition of nucleotide positions using the first nucleotide of the coding sequence of RefSeq NM_016124.4 (RHD isoform 1)

positive controls

No cDNA analysis was done for sample 2, because the cDNA sequence for this RHD deletion had been published before.

negative controls

None mentioned

Annotations

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