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Chapter 20: Introduction to Molecular Genetics

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One complicating factor in the process of DNA replication is the fact that the two strands of DNA are antiparallel to one another. DNA polymerase III can only catalyze DNA chain elongation in the 5 to 3 direction, yet the replication fork proceeds in one direction while both strands are replicated simultaneously. Another complication is the need for an RNA primer to serve as the starting point for DNA replication. As a result of these two obstacles, there are different mechanisms for replication of the two strands. One strand, called the leading strand, is replicated continuously. The opposite strand, called the lagging strand, is replicated discontinuously.

For the leading stand, a single RNA primer is produced at the replication origin and DNA polymerase III continuously catalyzes the addition of nucleotides in the 5 to 3 direction, beginning with addition of the first nucleotide to the RNA primer.

On the lagging strand, many RNA primers are produced as the replication fork proceeds along the molecule. DNA polymerase II catalyzes DNA chain elongation from each of these primers. When the new strand "bumps" into a previous one, synthesis stops at that site. Meanwhile, at the replication fork, a new primer is being synthesized by primase. The final steps of synthesis on the lagging strand involve removal of the primers, repair of the gaps, and sealing of the fragments into an intact strand of DNA. The enzyme DNA polymerase I catalyzes the removal of the RNA primer and its replacement with DNA nucleotides. In the final step of the process, the enzyme DNA ligase catalyzes the formation of a phosphodiester bond between the two adjacent fragments. It is little wonder that this is referred to as lagging strand replication.
A cloned piece of DNA is separated into its two strands. Each of these will serve as a template strand to carry out DNA replication in test tubes. A primer strand is also needed. This is a short piece of DNA that will hybridize to the template strand. The primer is the starting point for addition of new nucleotides during DNA synthesis.

The DNA is then placed in 4 test tubes with all of the enzymes and nucleotides for DNA synthesis. In addition, each tube contains an unusual nucleotide, called a dideoxynucleotide. These nucleotides differ from the standard nucleotides by having a hydrogen atom at the 3 position of the deoxyribose, rather than a hydroxyl group. When a dideoxynucleotide is incorporated into a growing DNA chain, it acts as a terminator. Because it does not have a 3OH group, no phosphoester bond can be formed with another nucleotide and no further polymerization can occur.

Each of the 4 tubes containing the DNA, enzymes, and an excess of the nucleotide required for replication will also have a small amount of 1 of 4 dideoxynucleotides. In the tube that receives dideoxyadenosine triphosphate (ddA), Dna synthesis will begin. As replication proceeds, either the standard nucleotide or ddA will be incorporated into the growing strand. Since the standar nucleotide is present in excess, the dideoxynucleotide will be incorporated infrequently/randomly. This produces a family of DNA fragments that terminate at the location of one of the deoxyadenosine in the molecule.

The same reaction is done with each of the dideoxynucleotides. The DNA fragments are then separated by gel electrophoresis on a DNA sequencing gel. The four reactions are placed in four wells, side by side, on the gel. Following electrophoresis, the DNA sequecne can be read directly from the gel:

When the chain termination DNA sequencing was first done, radioactive isotopes were used to label the DNA strands. Now automated systems that employ dideoxynucleotides that are labels with fluorescent dyes, a different color for each. Because each reaction (A, G, C, T) will be a different color, all the reactions can be done in a single reaction mixture and the products separated on a single lane of a sequencing gel. A computer then reads the gel by distinguishing the color of each DNA band. The sequence info is directly stored in a database for later

There is now a handheld DNA sequencer that can be used in environments from med lab to field. Cost-efficient and easy to use, they can determine the sequence of any DNA strand that enters a nanopore in the device. As each of the bases of the DNA strand passes through the nanopore, there is a distinctive change in an ionic current across the pore, by reading these changes, the device can read long stretches of DNA with 97% accuracy. The sequence readout is immediate.