Blog Against Cancer

NUCLEOTIDE SEQUENCING

The nucleotide sequence of a gene’s coding region encodes the amino acid sequence of its protein. This means that even in the absence of any knowledge about a gene’s protein, we can predict the structure of that protein given the nucleotide sequence of the gene. How can the nucleotide sequence of a gene be determined?

There are two methods used for sequencing DNA, the “chemical modification” method devised by Maxam and Gilbert,25 and the “enzymatic chain termination” method devised by Sanger and his colleagues.  Because of its ease and wider use, the chain termination method will be described here.
The chain termination method relies on properties of enzymes called “DNA polymerases” . These are enzymes that create new DNA polymers starting from individual nucleotides. However, in order for a DNA polymerase to work, it needs a “template” of single- stranded DNA on which to create the new polymer. DNA polymerase adds a new nucleotide to the 3′ end of a growing DNA chain, but the base of the new nucleotide must be able to base-pair (i.e., be complementary) to the base on the template over which the polymerase
is positioned. After the addition of that nucleotide, the polymerase moves to the next nucleotide on the template, and adds a new nucleotide to the 3′ end of the growing chain. Again, the new nucleotide must be complementary to the next base in the template.

When the process is completed, the DNA polymerase will have made a new DNA chain whose nucleotide sequence is completely complementary to the template DNA.
Nucleotide sequencing is based on the observation that when DNA polymerase adds a synthetic abnormal nucleotide to a growing chain, the polymerization stops. The synthetic “terminating” nucleotides used most commonly are dideoxynucleotides that have no alcohol substitutions on the 3′ carbon of their deoxyribose groups, and thus cannot be joined by a phosphate bridge to the next nucleotide . For example, in the presence of dideoxy-ATP (ddATP), chain termination will occur wherever an A appears in the new DNA sequence  These reactions are performed in vitro in a test tube, where millions of new DNA molecules are being made at once. If normal deoxy-ATP is mixed in the proper proportion with dideoxy-ATP, only a few of these molecules will terminate at each T in the template. This will generate a series of new DNA polymers,
each one stretching from the beginning of the chain to the position of an A . If the newly formed DNA is
radiolabeled, and the products of this reaction are separated electrophoretically in a polyacrylamide gel (see below), a ladder of radioactive bands will be generated. Each step of the ladder is a fragment of DNA that stretches from the start of the new polymer to the position of an A. Four separate reactions are performed using each of the four dideoxynucleotides. Each reaction is run in an adjacent lane on a polyacrylamide gel so that the nucleotide sequence can be read directly from the gel by reading up the steps of each ladder.
A specific application of DNA sequencing in cancer research has been the analysis of mutated sequences in the tumor suppressor gene p53.

The hallmark of tumor suppressor gene involvement in cancer is loss of function of these genes. While loss of function can occur by deletion of all or part of the gene, the same result can be achieved if the gene
undergoes a mutation that inactivates its protein. Thus, in many types of cancers that have retained a p53 allele, as determined by Southern blotting, DNA sequencing has shown that the remaining allele has often
undergone a single nucleotide, or “point,” mutation.27,28

RAPID TECHNIQUES FOR DETECTING MUTATIONS

Powerful as DNA sequencing may be, it is usually too cumbersome to be used as a screening tool for the identification of single mutations in patient DNA samples. A variety of clever techniques have been developed, which rapidly reveal single-base mutations without resorting to DNA sequencing.29 One is denaturing-gradient gel electrophoresis (DGGE), which depends on the fact that double-stranded DNA molecules “melt” or denature into single strands at different temperatures or chemical conditions, depending on their specific sequences.

For example, one can construct electrophoresis gels that contain a gradient of increasing concentrations of denaturants, such as urea or formamide, and if DNA is electrophoresed through such a gel, it will stop
migrating at the position at which it has denatured. If two DNA fragofments of identical length differ in their sequences at only one base pair, the concentration of denaturant at which the two fragments melt will
be slightly different. Thus, electrophoresis of these two DNA fragments through a gradient of denaturant will distinguish them by the positions at which the two fragments stop migrating. One could begin with fragments isolated by polymerase chain reaction (PCR) (see below), making this a convenient way to screen for the presence of common mutations using only a small amount of patient material.

Another simpler technique is single-stranded conformation polymorphism (SSCP), which relies on the differences in mobility between single-stranded DNA molecules on the basis of their secondary structures
in nondenaturing gels. Single-stranded DNA molecules can fold back on themselves due to intrastrand base-pairing and form unique shapes called “secondary structure.” Alteration of one base in a short
DNA molecule could, therefore, have profound effects on secondary structure by altering the pattern of intrastrand base-pairing. DNA molecules of identical length but different secondary structure will migrate at different rates in nondenaturing electrophoretic gels. Thus, DNA fragments can be isolated or synthesized by performing PCR on patient DNA samples, they can then be denatured, and individual strands allowed to reanneal to themselves rather than to their complementary strands. The products can be separated by nondenaturing electrophoresis, and fragments containing single base pair mutations can be identified by their anomalous migration. Although technically simpler than DGGE, which can detect nearly 100% of single base pair mutations, SSCP can only detect about 80% of such mutations.

POLYMERASE CHAIN REACTION

To detect gene sequences by Southern blotting, at least 1 to 2 μg of genomic DNA is required. This translates into milligram quantities of tissue that must be used fresh or freshly frozen. By amplifying specific fragments of DNA, the PCR lowers the theoretical limit of detectable DNA sequences in a sample to
a single molecule of DNA. With some advance knowledge of thenucleotide sequences in the DNA to be detected, microscopically small amounts of tissue, even a single cell, contains enough DNA to be
amplified, and the amplified DNA can be easily analyzed. Even fixed tissue in paraffin blocks or on slides can yield sufficient DNA for analysis using PCR.

The concepts underlying PCR are diagrammed in Figure 1.9. Two short single-stranded DNA fragments, called primers, have sequences complementary to those that flank the stretch of DNA to be amplified.
They are added to the target DNA, the mixture is heated to dissociate the paired double strands of target DNA, and then the temperature is lowered to permit hybridization, or annealing, of the primers to their
complementary sequences on the target DNA. A DNA polymerase enzyme is added to the mixture which will add nucleotides to the 3′ end of the primers using the target DNA as a sequence template. This step generates one copy of each of the strands of one target DNA molecule. The mixture is heated again to dissociate the strands, then cooled to allow more primers to anneal to the target sequences on both
the original and new pieces of DNA. DNA polymerase is added again and now generates four copies of the target sequences. These steps are repeated, resulting in a geometrically increasing amount of target
DNA, that is, a chain reaction.

When it was first devised, this technique used a DNA polymerase from E. coli, which is inactivated by heating, so that fresh enzyme had to be added at every step. With the discovery and cloning of
the DNA polymerase from the thermophilic bacterium, T. aquaticus (the Taq polymerase), which retains activity after being heated to 95°C, heating and cooling steps could be carried out on the same mixture
without adding new enzyme.35 This allowed the procedure to be automated. There are now automated thermal cyclers in every molecular biology laboratory, and in many clinical laboratories, that will take
PCR mixtures through 20 to 50 cycles, producing large amounts of synthetic DNA for subsequent analysis.