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The molecular biology laboratory is the starting point for most structural biology projects. These projects involve the study of protein molecules, most of which are present in the body only in minute quantities. The instructions for making proteins are found within the genes, the hereditary material that is composed of deoxyribonucleic acid (DNA), contained in the chromosomes present in each cell, and passed from one generation to the next. The main objective of the molecular biology lab is to manipulate the DNA that codes for a protein of interest in ways that make it possible to produce the large quantities of protein required for structural studies.
Identification of a DNA target: When the genetic instructions for making a particular protein are defective, the protein is likely to malfunction, resulting in disease. Proteins are chosen for study primarily because it is known that they are involved, in some way, with a disease process. In recent years, various genome projects have provided much new information about the DNA of humans and many other species. As a result, many new genes have been discovered. HWI scientists use the genomic information to identify genes, and their protein products, that are of interest for structural study.
Copying the target DNA: Once a suitable target gene has been identified, it is necessary to separate it from the rest of the genome. In higher organisms like humans, the entire DNA code for a single protein does not exist as one continuous piece. Instead, genes are broken into exon (coding) and intron (non-coding) regions. Cells are able to extract the coding regions by transcribing the DNA to form a single coding strand of messenger RNA (mRNA). In the molecular biology lab, the desired mRNA is first extracted from cells, and next the enzyme reverse transcriptase (RT) is used to generate a corresponding complete coding copy of the DNA (cDNA). This cDNA template can then be used in a procedure known as the polymerase chain reaction (PCR) to amplify or make many copies of the gene of interest.
Cloning the DNA: The next step involves the introduction of the PCR-amplified DNA into plasmids - naturally occurring small pieces of circular DNA that can replicate independently of the chromosomes. This is accomplished with the help of enzymes (restriction endonucleases) that cut DNA at specific sites and enzymes (ligases) that reattach the cut ends. The modified plasmids are called expression vectors, and they are optimized to overexpress (produce very large quantities) of the desired protein when they are introduced (transformed) into bacterial cells. Furthermore, many expression vectors are engineered to add an additional piece, called an expression tag, onto the protein molecule. Expression tags are used in subsequent protein purification steps to aid in the separation of the desired protein from contaminants.
Next Step: Protein Purification