HPLC: High-Performance Liquid Chromatography

A recent advancement of HPLC has been the development of the denaturing HPLC method (DHPLC). This procedure can separate DNA from different sources is placed into the column and treated to allow it to separate and recombine. Two strands from the same source, termed a homoduplex, bind strongly and tend to remain together longer. Heteroduplexes do not attract as strongly, and remainn unattached more often. double-stranded DNA molecules that differ by as little as one base pair. The speed of analysis (approximately 5 minutes per sample) and the size of DNA fragment that can be analyzed (up to 2.0 kilobytes) has made it a preferred method for a variety of applications in the field of molecular biology. Applications of DHPLC include the detection of single nucleotide polymorphisms (SNPs). These are single base-pair variations in DNA that can give valuable information on genetic variation within a population. They can also help to identify the genes that cause certain human diseases.

To determine if the two genes of interest differ, they are first amplified by the polymerase chain reaction and then injected together into a so-called reversed-phase column. In this type of column, the stationary phase is less polar than the mobile phase, which is the opposite of the arrangement found in standard columns. Once the genes are injected, their DNAs bind to the stationary phase. Increasing the temperature causes each gene to separate A high-performance liquid chromatography system allows geneticists to perform molecular research efficiently by automating necessary processes. into its two strands (a phenomenon called denaturing). Once denatured, the strands can enter the mobile phase and move through the column.

Cooling the column causes the strands of the genes to rejoin, and the DNAs reattach to the column. The temperature is manipulated to make the strands constantly separate and rejoin, with the balance determined by the strength of the attraction that exists between the strands. If the two genes are exactly identical, they will spend more time in the stationary phase, and elute from the column more slowly. If the genes differ even by a single nucleotide, however, they will spend more time in the mobile phase, and leave the column more quickly.

Y chromosome analysis is one of the most powerful molecular tools for tracing human evolution. Polymorphisms in the human Y chromosome, detected by DHPLC, can be used as markers for tracing human evolution. This will eventually help to elucidate the patterns of human origins, migration, and mixture. The ability to rapidly and efficiently genotype SNPs by use of DHPLC is also useful in medicine, through the identification of mutations that result in susceptibility to certain diseases or that affect physical responses to certain drugs.