The possibility of putting mass spectrometry technology to use in the Human Genome Project is now being explored by the instrumentation group at LBL's Human Genome Center. Led by physicist Joe Jaklevic and chemist Henry Benner, researchers in the group have constructed what they call a "time-of-flight mass spectrometry test stand" in which they will be testing different means of vaporizing and detecting molecules of DNA.
The Human Genome Project calls for mapping the location of genes on chromosomes and determining the sequences of the nucleotides that make up those genes. This entails breaking chromosomes into small fragments since even the smallest chromosome is too large to be handled intact with current technology. The resulting fragments must then be sorted according to size and this is perhaps the single most time-consuming step in the mapping and sequencing process.
The most widely used technique for sizing DNA fragments is called gel electrophoresis in which fragments are placed in a polymeric gel (such as agarose) and an electric field is applied. Because of DNA's negative electrical charge, the fragments move across the gel toward the positive electrode, with shorter fragments moving faster than longer fragments. Gel electrophoresis is sensitive enough to distinguish a size difference of only a single nucleotide base pair, but the process is slow because it takes so long for the fragments to transverse the gels. At the recent Department of Energy site review of LBL's Human Genome Center (see Currents, Jan. 17, 1992) one of the most enthusiastic responses from the site reviewers came during the opening report by center director Jasper Rine when he discussed a plan to test the potential use of mass spectrometry as an alternative to gel electrophoresis.
In a mass spectrometer, a molecular sample is vaporized and the resulting ions are separated according to their mass as they pass through a magnetic field. The ions are then directed into a detector for identification and analysis. Mass spectrometry data can be collected in less than one minute per sample -- substantially faster than the approximately four hours required to complete a gel electrophoresis run. However, past attempts at applying mass spectrometry to genome research were unsuccessful because no one could vaporize molecules as large as a typical DNA fragment.
"Recent progress in matrix-assisted laser desorption and plasma desorption has come close to solving the vaporization problem," says Benner. "Our calculations suggest that sequences up to 300 bases long should be analyzable with modest extensions of existing (mass spec) technology."
The time-of-flight mass spectrometry test stand that Benner and Jaklevic have constructed is designed to test and develop new ways of vaporizing and ionizing DNA fragments that are larger than 300 bases.
The most promising approach so far, they believe, is matrix- assisted laser desorption, which, potentially, could handle fragments as long as 2,000 base pairs. The matrix is an ultraviolet radiation-absorbing material that is mixed with the DNA fragments. When a pulsed UV laser zaps the mixture, the matrix absorbs the light's energy. This energy is transferred to the DNA, ionizing it and driving it down a time-of-flight tube and into a detector. Since velocity decreases with an increase in mass, the smaller fragments reach the detector first. Flight time is determined by counting the flashes of pulsed laser light.
Benner says there are limitations with the detectors now available that require repeated analysis of a sample to accumulate a useful mass spectrum when matrix-assisted laser desorption is used to ionize the DNA fragments. He and Jaklevic are also exploring the use of particle beam approaches, such as the electrospray technique in which the sample molecules (in this case, DNA fragments) are sprayed with electrically charged droplets. The mix is then evaporated, propelling the ionized DNA down the time-of-flight tube.
So far, ions generated by an electrospray possess a multiplicity of charge that makes the resulting mass spectra confusing and difficult to interpret. To overcome these problems, Benner and Jaklevic plan to test new types of ion detectors on macromolecules prepared by the biologists at the Human Genome Center. These new detectors will be based on either high voltage ion acceleration, secondary ion emission, or a combination of both.
Says Jaklevic, "We will also study advanced bolometric detectors which directly measure the thermal energy associated with ions."
Genome Center director Rine told the site reviewers that the challenge of sequencing by mass spectrometry is to be able to resolve the mass of, for example, the 300th base from the base at 299 with sufficient precision to know if the 300th base is guanine, adenine, thymine, or cytosine. Building upon the traditional strength of LBL in the field of detector design and development, he believes that researchers at the Human Genome Center will be able to use mass spectrometry to measure the inheritance of human genetic markers by the end of 1992.