Denaturing High Performance Liquid Chromatography

Denaturing high-performance liquid chromatography (DHPLC) is an ion-paired, reversed-phase, liquid chro-matography method used to identify mutations, including SNPs and small insertions or deletions, through its ability to separate heteroduplex DNA from homoduplex DNA47. DHPLC is conceptually similar to heteroduplex analysis (HA; see above). Conventional HA makes use of a gel matrix to separate homo- and heteroduplex species in a non-denaturing environment, whereas DHPLC uses partially denaturing conditions in a liquid chromatography column to exaggerate the separation between the two species.

The gene to be studied is first amplified using PCR. High-fidelity PCR is used to prevent the production of PCR artifacts (pseudoalleles) that could produce false-positive results. Optimal amplicon length is between 100 and 500bp. Purifying the PCR product is usually not necessary, as unincorporated primers, nucleotides, and genomic DNA do not interfere with the analysis. DHPLC requires het-eroduplex formation, accomplished by heating and slow cooling. Therefore, for conditions in which only one variant allele type may be present (such as recessive diseases, X-linked conditions in males, or small tumor samples with loss of heterozygosity in all cells), PCR products from normal and patient samples are mixed in equal proportions before heating and cooling to produce heteroduplex DNA and distinguish from homozygous wild-type alleles. The addition of normal PCR amplicons is not required when using DHPLC to test PCR products from heterozygous individuals, which naturally form heteroduplexes when denatured and slowly cooled.

The duplexes are injected into a DHPLC column, and the DNA binds to the stationary matrix. Binding is aided by triethylammonium acetate (TEAA). Because the stability of the binding depends on the temperature, the column is optimally held at the melting temperature of the PCR fragment. The melting temperature can be calculated using a variety of proprietary or free software programs. The DNA is next eluted using acetonitrile, an organic solution that facilitates the subsequent separation of the DNA from the column matrix, and DNA absorbancy is measured at 260 |im. The linear gradient of acetonitrile established in the column allows separation of DNA fragments based on size or the presence of heteroduplexes, or both. All DNA fragments impart a characteristic profile when the absorbance is plotted against elution time. The peak of maximum absorbance is the retention time of that DNA sample at a given acetonitrile concentration. Het-eroduplexes are less stable and thus have a lower affinity for the column. The concentration of acetonitrile required to separate heteroduplexes from the column is therefore lower, so heteroduplexes elute from the column earlier than homoduplexes.

The column temperature and gradient conditions can be optimized for the separation of any heteroduplex-homoduplex mixture. Some DNA fragments have more than one melting domain and the analysis may be carried out at more than one temperature. One advantage of DHPLC is that reinjection of the same sample at different temperatures is possible. Other advantages include high detection rates of mutations, rapid separation times per sample, a high degree of automation, and the ability to collect elution fractions and sequence each eluted fragment. Disadvantages of DHPLC include the need for expensive equipment and columns, high-fidelity PCR, and optimization of each reaction required to achieve the highest sensitivity of mutation detection.

Examples of Applications of DHPLC

1. RET and CFTR mutation detection48

2. BRCA1 and BRCA2 mutation analysis49

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