PCRRestriction Fragment Length Polymorphism RFLP

The most commonly used method to confirm genetically the diagnosis of SMA is a qualitative PCR-RFLP assay to detect the homozygous absence of SMN1.[22] The PCR-RFLP assay takes advantage of the base differences in exons 7 and 8 to distinguish SMN1 from SMN2. The restriction endonuclease Dral digests only the SMN2 exon 7 PCR products because of a restriction site generated by a mismatched reverse primer.[22] Another method introduces HinfI control restriction sites in both the SMN1 and the SMN2 exon 7 PCR products to ensure complete digestion, in addition to the SMN1 unique restriction site.[16]

PCR-Denaturing High-Performance Liquid Chromatography (DHPLC)

A rapid PCR-DHPLC method to detect homozygous absence of SMN1 exon 7 has been described.[23]

Prenatal and Preimplantation Genetic Testing

Prenatal testing to detect the homozygous absence of SMN1 can be performed on chorionic villous sampling (CVS) specimens, amniotic fluids, or maternal peripheral blood.[24] Preimplantation genetic testing to detect the homozygous absence of SMN1 exon 7 has been described (reviewed in Ref. [10]).

Quantitative SMN Gene Dosage Analysis

To identify SMA carriers necessitates a quantitative approach, i.e., SMN gene dosage analysis. The first highly precise assay to determine copy numbers of SMN1 and SMN2 utilizes quantitative competitive PCR-RFLP, with CFTR exon 4 as a two-copy genomic reference.[13] To correct for variation in amplification efficiency, SMN and CFTR internal standards are used. Each standard has the same primer binding sites as its genomic counterpart, and a small internal deletion, which allows the internal-standard PCR products and the genomic PCR products to be separated and quantified independently.1-13-1 The rationale for this quantification method has been described.[25] Various dosage analysis methods have since been described,[14,16,26,27] including real-time quantitative PCR.[15,28] SMN gene dosage analysis can be used for the detection of heterozygous absence of SMN1 in SMA patients who do not lack both copies of SMN1.[16]

Heteroduplex formation between SMN1 and SMN2 PCR products may affect quantitative PCR-RFLP.[25] In addition to heteroduplex formation, reproducible PCR bias between SMN1 and SMN2 sequences may be present in SMN gene dosage analysis.[29]

SMN1 Small Intragenic Mutation Analysis

Analysis for the detection of small intragenic mutations is typically performed by DNA sequencing. Although labor-intensive and not routinely offered, it can be performed.[20] A multiplex, allele-specific PCR assay to detect the seven relatively common small intragenic mutations has been described.[30]

Monosomal Analysis

A monosomal-analysis method utilizes the separation of single human chromosomes of homologous pairs by fusing human cells with mouse cells and culturing with selection. A monosomal-analysis method to identify an SMN1 deletion/conversion carrier who had two copies of SMN1 on the normal chromosome 5 (the ''2+0'' genotype) has been developed.[31] This promising technology obviates the need to perform extensive linkage and dosage analyses to distinguish carriers with two copies of SMN1 from noncarriers (the ''1 + 1'' genotype) who passed a de novo deletion mutation to an affected child.[3]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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