IV.

Mapping and Sequencing

There are two main categories of gene-mapping techniques: linkage, or genetic, mapping, a method that identifies only the relative order of genes along a chromosome; and physical mapping, more precise methods that can place genes at specific distances from one another on a chromosome. Both types of mapping use markers in the DNA sequence, detectable physical or molecular characteristics that differ among individuals and that are passed from one generation to the next.

Linkage mapping was developed in the early 1900s by American geneticist Thomas Hunt Morgan. By observing how frequently certain characteristics were inherited in combination in numerous generations of fruit flies, he concluded that traits that were often inherited in combination must be associated with genes that were near one another on the chromosome. From his studies, Morgan was able to create a rough map showing the relative order of these associated genes on the chromosomes, and in 1933 he was awarded the Nobel Prize for physiology or medicine for his work.

Human linkage maps are created mainly by following inheritance patterns in large families over many generations. Originally, these studies were limited to inherited physical traits that could be observed easily in each family member. Today, however, sophisticated laboratory techniques allow researchers to create more detailed linkage maps by comparing the position of the target gene relative to the order of genetic markers, or specific known segments of DNA.

Physical mapping determines the physical distance between landmarks on the chromosomes. The most precise physical mapping techniques combine robotics, lasers, and computers to measure the distance between genetic markers. For these maps, DNA is extracted from human chromosomes and randomly broken into many pieces. The DNA fragments are then duplicated numerous times in the laboratory so that the resulting identical copies, called clones, can be tested individually for the presence or absence of specific genetic landmarks. Those clones that share several landmarks are likely to come from overlapping segments of the chromosome. The overlapping regions of the clones can then be compared to determine the overall order of the landmarks along the chromosome and the exact sequence in which the cloned pieces of DNA originally existed in the chromosome.

Very detailed physical maps that indicate the precise order of cloned pieces of a chromosome are usually required to determine the actual sequence of nucleotides. The Human Genome Project most commonly used the DNA sequencing method developed by British biochemist and two-time Nobel laureate Frederick Sanger. In Sanger’s method, specific pieces of DNA are replicated and modified so that each ends in a fluorescent form of one of the four nucleotides. In modern automated DNA sequencers, pioneered by American molecular biologist Leroy E. Hood, the modified nucleotide at the end of such a chain is detected with a laser, and the exact number of nucleotides in the chain is determined. This information is then combined by computer to reconstruct the sequence of base pairs in the original DNA molecule.

Duplicating DNA accurately and quickly is of critical importance to both mapping and sequencing. Scientists first replicated fragments of human DNA by cloning them in single-celled organisms that divide rapidly, such as bacteria or yeast. This technique can be time consuming and labor intensive. In the late 1980s, however, a revolutionary method of reproducing DNA, known as the polymerase chain reaction (PCR), came into widespread use. PCR is easily automated and can copy a single molecule of DNA many millions of times in a few hours. In 1993 American biochemist Kary Mullis was awarded the Nobel Prize for chemistry for developing this technique.

American molecular biologist J. Craig Venter, founder of Celera Genomics, and American molecular biologist Hamilton O. Smith developed an alternative approach that determines and assembles the sequence of entire genomes without the laborious and time-consuming physical mapping phase. Since 1995 their approach has been used to sequence the genomes of a number of bacteria, the fruit fly Drosophila melanogaster, and the human genome.