Genetic Protocols

Genomic DNA provides the blueprint for cells to organize into living organisms. DNA contains four repeating nitrogenous bases called adenine, guanine, cytosine and thymine. Researchers have successfully determined the entire base sequence, the genome, for many complex organisms including humans. There are many important reasons to sequence a genome. Medical genetics is concerned with discovery of genetic abnormalities causing diseases such as diabetes, Alzheimer's and cancer. Evolutionary geneticists compare genomes from related species to develop accurate taxonomy. Basic genetic protocols in research are the same no matter the purpose of research.

Isolating DNA from Cells

• Genetic research requires extraction of a pure DNA sample from cells. A basic protocol begins by disrupting and removing protective plant cell walls or animal cell membranes with an enzyme called lysozyme. Non-genetic cell components in the form of precipitates are removed by rapid spinning with a centrifuge while DNA remains in solution. DNA is then precipitated in the presence of sodium acetate salt with ethanol. Final centrifugation precipitates and pellets pure genetic material for research purposes.

Polymerase Chain Reaction

• Genetic research requires sufficient copies of specified DNA regions. The Polymerase Chain Reaction, or PCR, is a method for exponentially producing copies of DNA. The protocol requires purified DNA, Taq polymerase, bases called deoxynucleotides and small DNA strands to prime the designated area. The reaction mixture is placed in a thermocycler, an instrument capable of rapid temperature changes. Heating the mixture to 95 degrees Celsius separates double stranded DNA, then cooled to 48 degrees so primers can sit, or anneal, to matching bases. The temperature is increased to 72 degrees when Taq polymerase assembles deoxynucleotides, called extension, into a new copy of the DNA.

Genetic DNA Sequencing

• Sequencing data appears as colored bands designating DNA bases.

  Automated sequencing is used to determine exact base sequence and compare normal with abnormal DNA. The protocol adds fluorescent-labeled dideoxynucleotides, or ddNTPs, to normal PCR reactions, which randomly stops extension of DNA fragments. The products are DNA fragments different by a single base. Each base type has a different color. The final products are loaded on an automated sequencer, an instrument designed to separate fragments by size on a polymer using electric current. When the fragments reach the polymer endpoint, a digital camera records the base color and sends the data to computer for analysis.

Genotyping and DNA Fingerprinting

• DNA fingerprinting is used to identify human remains.

  Genotyping is a method that compares small segments of a genome from one organism to another. The goal is to detect small differences in PCR fragment size resulting from normal or accidental base changes in DNA. The protocol design begins with basic PCR using one fluorescent-labeled primer for detection on automated sequencers. Labeled products are separated by size and recorded by digital camera to a computer for analysis. Genotyping protocols are designed for forensic identification, DNA fingerprinting and paternity or identification of genetic abnormalities that result in disease.