Khorana laid the groundwork for chemical synthesis of DNA. He developed techniques to form the phosphodiester bond between nucleotides while at the same time preventing the reactive amino, hydroxyl, and other phosphorus groups from reacting. With these techniques, he and his co-workers then synthesized a complete tRNA gene. Originally many person-years were required for the synthesis of 80 nucleotide oligomers. Now, as a result of continued development by many research groups, oligonucleotide synthesis has been highly automated and as many as 100 nucleotides may be joined in specific sequence in a day.

In chemical DNA synthesis, blocking groups are placed on the reactive groups that are not to participate in the condensation to form a phosphodiester. These are then condensed to build the oligonucleotide (Fig. 1). After synthesis of the complete oligonucleotide, all the blocking groups are removed. If the desired oligonucleotideis particularly long, blocks of short, overlapping oligonucleotides can be synthesized, hybridized, ligated, and finally cloned.

Fig1. Outline of one method for the chemical synthesis of DNA. Note that elongation proceeds from the 3’ to the 5’ end in this method.

Before 1965, no researchers would have had a good idea of what to do with the sequence of an entire chromosome if it were presented to them. We are hardly in that situation now. Similarly, before 1975 there seemed to be little reason to try to synthesize DNA chemically. Not only were relatively few interesting sequences known, but the fraction of the synthesized material that would possess the desired sequence was likely to be too small to be of use. With the development of cloning since 1975 and the overall increase in our knowledge of biological mechanisms, the picture dramatically changed. Now it is routine to synthesize a gene de novo. Convenient restriction sites can be placed through the gene and when necessary, portions of the gene can be altered by synthesis of just the region between two restriction enzyme cleavage sites.

Another method for mutating a gene is to direct mutations to a specific point. This can be done with chemically synthesized oligonucleotides in a process called oligonucleotide directed mutagenesis. An oligonucleotide containing the desired alteration, a mutation, insertion, or deletion, will hybridize to complementary, wild-type single-stranded DNA and can serve as a primer for DNA pol I (Fig. 2). The resulting double-stranded DNA contains one wild-type strand and one mutant strand. Upon replication in cells, one of the daughter duplexes is wild-type and the other is mutant. Sometimes it is necessary to prevent heteroduplex repair of the mutant strand. Either way, following trans formation and segregation, a mutant gene can be obtained.

Fig2. Mutagenesis with chemically synthesized DNA. The oligomer hybridizes, except for the mispaired base. Extension of the primer with DNA Pol I and ligation yields heteroduplex molecules that can be transformed. Following DNA replication in the transformants, the two types of DNA molecules segregate to yield wild-type or mutant homoduplexes. Retransformation yields colonies containing entirely mutant or wild-type DNA, which can be identified by hybridization with radioactive mutant oligomer.

مواضيع ذات صلة

The Role of Inducer Analogs in the Study of the lac Operon

Background of the lac Operon

Altering Cloned DNA by in vitro Mutagenesis

Footprinting, Premodification and Missing Contact Probing

Megabase Sequencing

DNA Fingerprinting—Forensics

Isolation of Rare Sequences Utilizing PCR

Polymerase Chain Reaction

Southern, Northern, and Western Transfers

Finding Clones Using Antibodies Against a Protein

Finding Clones from a Known Amino Acid Sequence

Enzymatic DNA Sequencing