DNA & Biotechnology Notes
PreAP Biology
With the determination of DNA structure credited to Watson & Crick, and our subsequent understanding of its primary functions in cell reproduction (DNA replication) & gene expression (we’ll talk about this later this semester), scientists began to explore how this information could be exploited for the benefit of humanity. While some of the innovations involving the human genome are shrouded in controversy, some, such as gel electrophoresis and polymerase chain reaction (PCR), have been proven extremely valuable.
Old tools available for the diagnosis of genetic disorders include karyotypes, pedigrees, amniocentesis, chorionic villi sampling, ultrasound, and blood tests. These tools are still used, but science has gone way beyond just diagnosing a disorder! Scientists are looking toward treatments and cures, as well as enhancement and prevention of many genetic traits in the Animal Kingdom as well as all 5 of the other kingdoms. We will talk more about these following our unit on viruses and bacteria in the spring semester.
Gene technology really took off with the Human Genome Project. Initially a government venture with the goal of sequencing the entire human genome (all 23 different chromosomes), it soon turned into a contest between a privately funded team headed up by J. Craig Venter and the government team. The competition turned out to be just the spark needed. The project wrapped up in 2003. Nature published a historic article on this contest (which I believe was called a draw) in 2006. The methods used below are partially due to discoveries and innovations used during this historic quest for knowledge.
Vocabulary Basics
Biotechnology – manipulation of organisms or their components to make useful products
Gene technology is a generic term covering many new techniques involving DNA and/or chromosomes. These new techniques include genetic engineering, electrophoresis, Southern blots, DNA fingerprinting, and polymerase chain reaction (PCR).
Genetic engineering – direct manipulation of genes for practical purposes
Bioinformatics - application of computer science and information technology to the field of biology and medicine
Proteomics - term in the study of genetics which refers to all the proteins expressed by a genome
Genomics – study of whole sets of genes and their interactions once we have the entire genome sequence
Electrophoresis
Electrophoresis is a tool used by many different kinds of scientists to separate molecules based on characteristics such as molecular size, weight, or charge. The applications of electrophoresis in gene technology involve separating pieces of DNA (that have been cut by restriction enzymes) by size and making a Southern blot or a DNA fingerprint. Other techniques involve the separation of proteins using similar, electrophoretic materials.
The DNA is put into a solution and squirted into a small well at one end of the gel (literally a piece of gelatin-like material that is made of a seaweed extract plus other materials) in the electrophoresis chamber. The chamber is turned on and a current passes through the chemicals inside. The DNA pieces will move toward the positive pole (because the phosphates give DNA a negative charge). The small pieces will move the fastest.
(Here’s an easy way to think about why the small pieces move fastest. The gel is made of molecules that can get in the way of the DNA moving. The smaller the piece, the easier it is to move past the gel molecules, just like it is easier for you to walk alone between the trees in a dense forest than it is for you to walk between the trees with five friends all holding hands.)
DNA Fingerprinting
DNA fingerprinting is so named because scientists have realized that a person’s DNA is as unique as their fingerprint, therefore when restriction enzymes are used to cut a person’s DNA into pieces, the number and size of the pieces is unique and creates a unique banding pattern on the electrophoresis gel. The different pieces that result are called restriction fragment length polymorphisms (RFLPs). Polymorphism means “many forms” and refers to the fact that the fragments are different lengths.
DNA fingerprinting is used to determine guilt or innocence (or more accurately, whether or not someone was present where a crime occurred) using crime scene (forensic) material, as well as paternity, identity, and even the presence or absence of a specific genotype, which can aid in the diagnosis of a specific genetic disorder.
At a crime scene involving a murder, police recovered skin cells under the victim’s fingernails. The DNA in the skin cells was analyzed- it was not the victim’s, and thus was presumed to be the assailant’s. Three suspects were arrested for matching the description given by several witnesses. Their individual DNA was analyzed, and the results are shown below. Can the correct suspect be determined?
Southern Blot
A Southern blot (named after the scientist who developed the technique) is used to identify specific sequences of DNA that a scientist is interested in, such as determining which pieces have the insulin gene(p. 231, Figure 5).
DNA is run through an electrophoresis chamber and a piece of filter paper is laid on top of the gel to make an exact copy of the banding pattern. The filter paper is soaked in a solution containing radioactive nucleic acid pieces that are known to be complementary to the gene the scientist is looking for. The radioactive pieces will attach to the gene sought and that band will glow, indicating that what you are looking for is present in that sample.
A little more – Southern blots can be used to detect and analyze differences in banding patterns – these differences result from differences in DNA sequences on similar chromosomes from individual to individual or species to species. These differences, which are cut at different places by restriction enzymes, are called restriction fragment length polymorphisms or RFLPs (pronounced “rif-lips”)
Polymerase Chain Reaction (PCR)
Polymerase chain reaction is a method used to rapidly duplicate DNA segments – on the order of 1 billion copies per hour! The process mimics DNA replication. First, high temperature is used to separate the strands of the DNA. Then, low temperature and bacterial DNA polymerase are used to make copies (allow the free nucleotides in the solution to copy the DNA strands that have been separated. The machine then cycles to high temperature again to start the separation of the strands again. This continues over and over in a repeating cycle. Important applications include any situation where you have limited DNA – crime scene material, diagnosing a genetic disorder, studying a fossil, etc.