DNA Technology and Genomics

Introduction to Genetic Engineering

Genetic engineering involves the direct manipulation of an organism's genome.
The structure of DNA, deoxyribonucleic acid, the organic molecule containing coded instruction for the life processes of an organism, was discovered in 1953. Today, scientists are able to use their knowledge of DNA in the science of genetic engineering, the direct modification of a genome to change an organism's traits). Concepts that were once too unrealistic—designer DNA and altered DNA for the purpose of curing diseases, solving world hunger, and building useful organisms—are now actually in use or being studied.

The practical use of organisms or their products for the benefit of humans, or biotechnology, is not a new science. For centuries, humans have been domesticating animals for work, food, and companionship and cultivating plant crops for desirable traits. These practices change the genome (genetic material of an organism) to suit human interests. Traditionally, this process involved selective breeding instead of direct changes to DNA. The ability to directly modify DNA to give an organism a desirable trait that may not be possible through natural or artificial selection was developed only a few decades ago.

Genetic engineering involves the introduction of foreign DNA into an organism's genome, which is the entire set of DNA within an organism. This process results in recombinant DNA, which is DNA created from more than one individual, possibly of a different species. The organism whose genome was modified through the addition of recombinant DNA is called a genetically modified organism (GMO). All living organisms use the same genetic triplet code, which is the basis for being able to transfer DNA from one organism to another. Because all DNA has the same chemical properties, recombinant DNA can be made from organisms of any species. When an organism contains recombinant DNA made using genes from a different species, that organism is considered to be transgenic.

Making Recombinant DNA

Transferring genes from one organism to another makes recombinant DNA, which can produce traits that the organism does not naturally possess.
DNA carries the genetic code required by an organism to make proteins within its cells. As the cell makes a protein, it copies the instructions from the DNA and transcribes them to a new ribonucleic acid (RNA) molecule. These RNA molecules carry the instruction set from the nucleus to the cell's ribosomes. Genetic engineering allows scientists to insert DNA into an organism that contains the genetic code for a protein not normally made by the organism. Recombinant DNA technology is widely used in medicine and agriculture. For instance, a gene for insect resistance can be inserted into a crop plant that is normally affected by insects. The new transgenic crop plant will produce recombinant proteins (proteins made by an organism that contains a recombinant gene) associated with the inserted gene, which allows the transgenic crop to show more resistance to insects. However, encouraging the transgenic organism to express the inserted gene can be complicated because other components that assist in the decoding must also be added. This is especially problematic when trying to get a prokaryote (an organism without a nucleus) to express a eukaryotic (an organism with a nucleus) gene.

Once the new gene is incorporated into an organism's genome, the organism's own machinery for DNA replication will automatically replicate the gene, along with the rest of its DNA. In this way, recombinant DNA technology can be used to quickly mass-produce human, other animal, or plant genes (or proteins) inside naturally dividing bacterial cells. Bacteria are often used for this purpose because they are small, have relatively simple genomes, and reproduce quickly by cell division, making exact clones. Also, bacteria can naturally transfer a plasmid, a small, circular piece of bacterial DNA that replicates on its own and can be transferred between cells. These pieces of DNA are easily incorporated into other bacterial cells, making them the perfect DNA transportation vector (a carrier of recombinant DNA). Most human insulin currently available to diabetic patients in the United States is made using bacteria.

As an example of this process, assume protein A is naturally produced by a plant. This protein is believed to have potential medical applications. In order to further study it, scientists will need large amounts of the gene and the protein. One way to accomplish this is through DNA cloning, the use of recombinant DNA technology to replicate DNA within a rapidly reproducing organism. First, scientists insert gene A, which contains the genetic code for protein A, into a bacterial cell. Then, as the bacteria quickly divide, the gene will be multiplied, too. Eventually, entire bacterial colonies will carry gene A. The gene copies, or clones, can be harvested from the bacteria and used for research or other applications. Obtaining the protein itself is more complicated. Bacteria do not naturally carry the machinery for the proper translation (a process where ribosomes make proteins) and processing of eukaryotic proteins. However, bacteria may be coaxed into making protein A through the use of a promoter, such as a specific chemical or a certain temperature.
Bacterial plasmid (circular piece of bacterial DNA) and piece of host DNA containing the desired gene are both cut with restriction enzymes (proteins that cut DNA) and then combined to make recombinant DNA. Recombinant DNA is incorporated into bacteria and replicated as the bacteria reproduce. Gene clones may be collected, or the protein may be synthesized if bacteria have translation machinery (cellular structures that produce proteins).

Restriction Enzymes and Recombinant DNA

Restriction enzymes cut any piece of DNA at the same restriction sites, allowing unrelated DNA pieces to be connected at the "sticky ends."
DNA consists of six molecular building blocks, including a five-carbon sugar (deoxyribose), a phosphate, and four different nitrogenous bases. The nitrogenous bases are adenine, thymine, cytosine, and guanine, which are typically represented as A, T, C, and G, respectively. These are arranged in different sequences and paired in specific ways to make the DNA that is unique to each organism.

Special enzymes (catalysts) are used when making recombinant DNA. A restriction enzyme, or endonuclease, cuts DNA at a specific sequence point. A given endonuclease will cut any strand of DNA at the same sequence. Each location where a given piece of DNA has the recognized sequence is called a restriction site. When exposed to a particular restriction enzyme, all restriction sites will be cut. Most endonucleases do not cut straight through both strands of DNA in the same place; they make a jagged cut based on complementary base pairing (the alignment of nucleotide bases pair according to specific rules: adenine always pairs with thymine (or uracil in RNA), and cytosine always pairs with guanine).

A common endonuclease used in genetic modification is EcoRI (an enzyme isolated from a species of Escherichia coli bacterium). EcoRI always cuts between the G and A in the sequence GAATTC. Because of the complementary nature of double-stranded DNA, EcoRI cuts the DNA such that four nucleotide bases (two Ts and two As) will be "hanging" off the end of each piece, following a cut. These are called "sticky ends" because the locations that form hydrogen bonds are exposed and will tend to bind with other exposed complementary ends. All DNA fragments cut by this enzyme are left with the same sticky ends. So, any sticky end is equally likely to bind with any other. This second end may come from a plasmid (a circular piece of bacterial DNA), another piece of DNA, or the very same piece of DNA from which it was just cut. As a result, there are many different strands made when using restriction enzymes, and a few contain the gene that the scientist is attempting to isolate.
Restriction enzymes cut all strands of DNA at any location holding a specific sequence. The cut is jagged, forming two "sticky ends" that can be joined with any other complementary sticky end, that is, an end with a sequence that is the opposite of the sticky end.
Once a sticky end finds a complementary sticky end, the two ends will weakly bind together. Another enzyme, DNA ligase (an enzyme that assists in joining DNA strands), is used to strengthen the bond and complete the formation of the recombinant plasmid. Now, the plasmids are ready to be taken up by the bacterial cell to finish the transformation into the modified DNA.

There are several steps required for making recombinant DNA. These include:

Step 1. Isolate a plasmid (circular piece of bacterial DNA) from a bacterial cell and DNA from a plant cell.

Step 2. Cut the plant DNA and the bacterial plasmid using the same restriction enzyme (endonuclease).

Step 3. Mix together the plant DNA and bacterial plasmid fragments. Add DNA ligase (an enzyme for binding DNA) to bind the sticky ends together.

Step 4. Reintroduce the recombinant plasmids to bacterial cells and encourage the bacterial cells to take the new plasmids. The uptake and incorporation of foreign DNA by a bacterial cell is called transformation.

Step 5. Isolate the bacterial clones that contain the desired gene and allow them to reproduce.

Step 6. Harvest copies of the desired gene.

Step 7. Make the necessary modifications for protein synthesis in a vector.

Step 8. Collect the desired protein.