Gene Patents (Part 3 – the Science, continued)
In Part 2 of this series, we looked at one type of DNA, complementary DNA, also known as cDNA, in terms of its basic structure. cDNA has many uses in molecular biology. Because the sequence of nucleotides in a molecule of cDNA is the same as that in the genomic DNA, cDNA can be sequenced to determine that nucleotide sequence. This is possible because cDNA is synthesized from the messenger RNA (mRNA) that is made in the cell by transcription from the gene or open reading frame. The cDNA will have only the exons (the coding portion) and not the introns (the non-coding portion) of the gene.
In this part, we will look at the standard molecular biology tools that can be used to create and manipulate DNA, and particularly cDNA.
In eukaryotic cells (i.e., those cells that have a nucleus), genomic DNA is packaged into the chromosomes. Human beings have 23 pairs of chromosomes. Each chromosome contains billions of nucleotides in the DNA. Standard techniques have been developed for isolating the genes or open reading frames in this DNA and determining the nucleotide sequence. Space does not permit a detailed study of these techniques, but we can discuss the general idea.
In order to obtain enough DNA to sequence, the candidate DNA must be “amplified.” Sequencing uses automated machines that analyze the DNA chemically. Although these machines can work with very small quantities of DNA, there is a minimum amount. Eukaryotic cells (e.g., human tumor cells) express many genes as mRNA. Only a fraction of the total mRNA in a cell will be the mRNA that is expressed by the gene of interest. Eukaryotic cells divide relatively slowly, and it would take a very long time, even if the cells of interest were grown outside the body, to obtain enough cells to extract the DNA of interest.
Instead, the entire eukaryotic genomic DNA may be digested into smaller segments by “restriction enzymes.” These enzymes break the covalent bonds (see Part 2) between the nucleotides only at certain nucleotide sequences. If the genomic DNA is partially digested by a restriction enzyme, the size distribution of the resulting restriction segments will depend upon how many nucleotides are in the recognition sequence for that particular restriction enzyme. By using the correct restriction enzyme (which are commercially available) and adjusing the conditions of the enzymatic reaction, a desired restriction segment size may be obtained. It will then be possible to obtain a mix of restriction segments containing all of the nucleotide sequences in the genomic DNA, including that for the target gene. However, there may not be enough DNA in each restriction segment to sequence.
The next step is to clone the restriction segments into an organism, such as the bacterium E. coli, whose cells divide very rapidly. Cloning is generally done by inserting each restriction segment into small segments of the bacterial DNA called “plasmids” that replicate within the bacterial cell.
Commercially available plasmids contain multiple restriction sites (corresponding to the restriction site that was used to digest the eukaryotic DNA, and others), some sort of selection site (e.g., a gene that gives the bacterial cell resistance to a particular antibiotic), and a “marker” for the detection of the clones. Plasmids such as these are called “vectors.” One commercially available vector, call pUC19, is shown below. The site amp confers resistance to the antibiotic ampicillin. The site lacZa is a marker for the enzyme beta galactosidase. The “polylinker” is a segment of plasmid DNA that contains several restriction sites, including the restriction site for the restriction enzyme that was used to digest the eukaryotic DNA.
If the partially digested DNA from the eukaryotic cells is mixed with a digest of the plasmid, and the bacterial cells are cultured in a growth medium, a certain number of the bacterial cells will contain a plasmid that contains the restriction segment that in turn contains the genomic DNA of interest. These cells can than be plated out onto a growth medium that contains the antibiotic, and only those bacterial cells that contain the plasmid with the gene that gives resistance to the antibiotic will grow. Further selection can be done by using a marker, such as a gene that codes for the enzyme beta galactosidase. This gene, if intact in the cell, will give the cell a white color, and if the marker gene is not intact, the cell will have a blue color. If a restriction segment is inserted into the polylinker, the lacZ site will not be intact. Colonies of bacteria that are white will then each contain a plasmid that includes a restriction segment. Each colony is then grown separately in culture, resulting in separate batches of cells for each restriction segment. The DNA is then extracted from each batch of cells and used to produce cDNA by the reverse transcriptase enzyme discussed in Part 2 of this series. Each restriction segment from the original eukaryotic DNA will then be represented by a cDNA molecule. The collection of such cDNA molecules is called a “library.” If the eukaryotic DNA was extracted from a tissue that would be expected to be enriched for the DNA of interest (if, for example, the DNA was extracted from a tumor and we were looking for the DNA that encodes a protein that is implicated in the tumor), then the cDNA is called a “shelf” in the library.
The restriction enzyme/vector method of amplifying cDNA involves the breaking of the covalent phosphodiester bonds in the polynucleotide chain. A different method, polymerase chain reaction (PCR), involves the breaking and re-forming of the hydrogen bonds between the two polynucleotide chains. Unlike the covalent bonds in the backbone of the DNA strands, the hydrogen bonds between the two strands can be denatured by heating the DNA, and can then be re-formed by cooling the DNA. If we know part of the polynucleotide sequence of the DNA of interest, we can incorporate a polynucleotide having this sequence and the individual nucleotides (A, T, G and C) in a reaction mixture with a DNA polymerase enzyme. The polynucleotide is called a “primer.” The mixture is heated to denature the DNA, the mixture is cooled to anneal the primer to each strand of the denatured DNA, and the DNA polymerase synthesizes a matching strand to each strand of the DNA. Then the new double-stranded DNA molecules are denatured, cooled, and annealed to the primer again. This cycle continues many times until a large number of copies of the original DNA have been synthesized. This can be done automatically by machines. The following figure shows the steps of PCR.
Once there is enough pure DNA, either through cloning or PCR, or both, standard biochemical techniques can be used to determine the sequence of the nucleotides in the DNA. These techniques are so standardized that it is not necessary to explore them in greater depth for the purpose of this series of articles.