Human Genome Project
DNA is the molecule that is the material for genes in all forms of life. Human DNA is being completely mapped in a massive international effort called the Human Genome Project. The project was originally planned to be completed in 2005,
but scientific advances have put it ahead of schedule with completion now
expected in 2003 or earlier. This is significant for preventive health
care because understanding the human genetic code will provide anti-aging and health remedies far beyond the significant therapies that are already available.
Each and every one of the trillions of cells in the human body contains a two meter length of DNA which includes the following in each cell: 46 human chromosomes (23 pairs), 3 billion DNA subunits (the bases A, T, C, and G), 80,000 genes that code for proteins that perform all life functions. The Human Genome Project will decode every one of the 3 billion subunits!
Some DNA details: Apart from reproductive gametes, each cell of the human body contains 23 pairs of chromosomes, each a packet of compressed and entwined DNA. Every strand of the DNA is a huge natural polymer of repeating nucleotide units, each of which comprises a phosphate group, a sugar (deoxyribose), and a base (either Adenine, Thymine, Cytosine, or Guanine). Every strand thus embodies a code of four characters (A's, T's, C's, and G's), the recipe for the machinery of human life. In its normal state, DNA takes the form of a highly regular double-stranded helix, the strands of which are linked by hydrogen bonds between adenine and thymine (AÐT) and between cytosine and guanine (CÐG). Each such linkage is said to constitute a base pair; some three billion base pairs constitute the human genome. It is the specificity of these base-pair linkages that underlies the mechanism of DNA replication illustrated here. Each strand of the double helix serves as a template for the synthesis of a new strand, the nucleotide sequence of which is strictly determined. Replication thus produces twin daughter helices, each an exact replica of its sole parent.
Spelling out the answer: In the much-automated Sanger sequencing method, the single-stranded DNA to be sequenced is "primed" for replication with a short complementary strand at one end. This preparation is then divided into four batches, and each is treated with a different replication-halting nucleotide (depicted here with a diamond shape), together with the four "usual" nucleotides. Each replication reaction then proceeds until a reaction-terminating nucleotide is incorporated into the growing strand, whereupon replication stops. Thus, the "C" reaction produces new strands that terminate at positions corresponding to the G's in the strand being sequenced. (Note that when long strands are being sequenced the concentration of the reaction-terminating nucleotide must be carefully chosen, so that a "normal" C is usually paired with a G; otherwise, replication would typically stop with the first or second G.) Gel electrophoresis -- one lane per reaction mixture -- is then used to separate the replication products, from which the sequence of the original single strand can be inferred.
Another way of amplifying DNA is the polymerase chain reaction, or PCR. American scientist Dr. Kary Mullis won the 1993 Nobel Prize for chemistry for the invention of this process. This enzymatic replication technique requires that initiators, or PCR primers, be attached as short complementary strands at the ends of the separated DNA fragments to be replicated. An enzyme then completes the synthesis of the complementary strands, thus doubling the amount of DNA originally present. Again and again, the strands can be separated and the polymerase reaction repeated -- so effectively, in fact, that DNA can be amplified by 100,000-fold in less than three hours. As with cloning vectors, the result is a large collection of copies of the original DNA fragment.
When a clone
library can be ordered -- that is, when the relative positions on the human
chromosomes can be established for all the fragments -- one then has the perfect
resource for achieving the project's central goal, sequencing the human genome.
How the sequencing is actually done can be illustrated by the most popular
method in current use, the Sanger procedure, which is depicted schematically
above. The first step is to prime each identical DNA strand in a preparation of
cloned fragments. The preparation is then divided into four portions, each of
which contains a different reaction-terminating nucleotide, together with the
usual reagents for replication. In one batch, the replication reaction always
produces complementary strands that end with A; in another, with G; and so on.
Gel electrophoresis is used to sift the resulting products according to size,
allowing one to infer the exact nucleotide sequence for the original DNA strand.
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