Info

aCD nomenclature: http://www.ncbi.nih.gov/prow/. Human gene nomenclature: http://www.gene.ucl.ac.uk/nomenclature.

aCD nomenclature: http://www.ncbi.nih.gov/prow/. Human gene nomenclature: http://www.gene.ucl.ac.uk/nomenclature.

Various HPAs have been genotyped by SSP-PCR.[4,5] More references are cited in Ref. [6]. Efforts have been made to standardize the PCR conditions for detecting different polymorphisms. A consensus protocol for genotyping of HPA-1 to -5 and HPA-15 can be found on the platelet immunology pages in the Haematology Division section of the National Institute for Biological Standards and Control web site: http://www.nibsc.ac.uk.

Restriction Fragment Length Polymorphism

Restriction fragment length polymorphism (RFLP) analysis has been implemented for genotyping of platelet antigens in many laboratories. This technique exploits the fact that single nucleotide substitutions may create or abolish a unique enzyme cleavage site. This makes possible allelic discrimination. Genomic DNA is first amplified by PCR using forward and reverse primers flanking the polymorphic region and then subjected to restriction enzyme digestion. The fragments are separated by electro-phoresis, stained and analyzed under UV light. The genotype is then interpreted from the restriction pattern.

Polymerase chain reaction-restriction fragment length polymorphism analysis is simple to perform but requires the extra post-PCR step of digestion of the amplified DNA fragment. However, digestion can be incomplete and results in a homozygous individual could be falsely interpreted as heterozygous. Therefore, to avoid undetected incomplete enzyme activity, the amplified DNA fragment should be designed with an additional site for the same restriction enzyme.

Literature for RFLP typing of many HPAs are listed in Ref. [6].

Allele-Specific Oligonucleotide Hybridization

Allele-specific oligonucleotide (ASO) hybridization has been used in several of the initial studies investigating the molecular basis of the HPA alloantigens. This technique utilizes a generic PCR amplification with primers flanking the specific SNP, immobilization on a nylon membrane, and hybridization to labeled allele-specific oligonucleo-tide probes. Under appropriate hybridization and wash conditions, these probes bind only to their complementary sequence and are able to distinguish single nucleotide differences. The PCR products bound to an allele-specific probe are then detected using a luminescent or chromo-genic substrate. Interpretation of the HPA types is based on the presence or absence of positive reactions. Allele-specific oligonucleotide hybridization has been used to detect various HPAs (references are cited in Ref. [6]).

A variation of the ASO technique is the reverse dot-blot hybridization.[7] The ASOs are covalently bound to a nylon membrane and the labeled amplification products are simultaneously hybridized to the probes.

Oligonucleotide Ligation Assay

In the oligonucleotide ligation assay (OLA), gene segments with the polymorphic sites are amplified in a single multiplex PCR. Aliquots of the PCR product are then incubated with pairs of labeled allele-specific probes complementary to contiguous sequences. One probe contains the allele-specific base. Where both probes are bound by complementary sequences, they can be co-valently linked by a thermostable DNA ligase to produce a double-labeled product. This double-labeled product can then be detected by an enzyme-linked im-munoabsorbent assay (ELISA). If the allele-specific base is absent from the template DNA, annealing of the allele-specific probe is incomplete and ligation cannot occur. No product can then be detected. Oligonucleotide ligation assay does not require the temperature-sensitive hybridization and washing steps used in the ASO technique. The ELISA makes the OLA system suitable for automation. HPA-1 to -5 have been typed with this system (reference in Ref. [8]).

Single-Strand Conformational Polymorphism

The mobility of single-strand DNA in nondenaturing polyacrylamide gels depends on size and tertiary conformation. Single-base changes alter the tertiary conformation such that the single-strand DNA shows a different mobility in the polyacrylamide gels. This is exploited in single-stranded conformational polymorphism (SSCP) to analyze HPA polymorphisms. The method has been used to genotype HPA-1 to -5 and HPA-10W (references are listed in Ref. [8]). The SSCP electrophoresis requires precise temperature control as any variation in temperature can affect single-strand DNA migration.

Preferential Homoduplex Formation Assay

The preferential homoduplex formation assay (PHFA) is based on DNA strand competition between a doublelabeled amplicon (a reference double-stranded DNA with each strand separately labeled) and the unlabeled DNA sample. During hybridization with a precisely controlled temperature gradient, preferential formation of a homo-duplex occurs more often than a heteroduplex. When reference DNA and test DNA are identical, heteroduplex formation is favored. If the sequences are not identical, there will be little reduction in the double-labeled population. The reformed double-labeled DNA is immobilized on a microtiter well and detected by ELISA system (for references, see Ref. [8]).

then emitted. Although developed for quantitative realtime PCR, TaqMan technique is suitable for high-throughput SNP analysis. For the biallelic HPA system, two allele-specific probes with two reporter labels, which can be differentiated spectrally, are added into the same PCR mix, allowing single-tube genotyping. References for HPA-1-3 typing are listed in Ref. [8].

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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