Chapter 12 Notes

Much of biotechnology today is based on our study and knowledge of bacterial genetics. In addition to reproducing through binary fission, bacteria may also undergo other means of genetic recombination. These include transformation, transduction, and conjugation. In each case, the recipient cell receives extra DNA that may encode useful genes. Plasmids are extra-chromosomal circular pieces of DNA that carry genes for useful traits like pilus production (for conjugation) and antibiotic resistance.

Because plasmids may carry genes for proteins that break down antibiotics, their bacterial hosts are then resistant to that antibiotic. Furthermore, those resistance plasmids can be passed to other recipient bacteria and that is how antibiotic resistance spreads. This has become a significant problem in medicine, because many pathogenic bacteria are now resistant to the drugs that have previously been useful in their treatment. The plasmids that are involved in conjugation are widely used in research and biotechnology because they can be ‘engineered’ to contain certain genes. Along with antibiotic resistance genes, other genes can be cloned into plasmids and their presence can be tested with the engineered plasmids are ‘transformed’ into recipient cells.

The method used to insert new genes into a plasmid involves the use of a group of enzymes that cut DNA at specific nucleotide sequences. These enzymes are called restriciton endonucleases and the sequences that they recognize in DNA are called palindromes because the sequence is the same forwards as backwards. For example, the statement, "Madam I’m Adam" is a palindrome. Frequently, restriction endonucleases cut DNA and leave overhanging nucleotides on one strand of the DNA. These are called ‘sticky ends’ and they play an important part in engineering DNA. Other segments of DNA can be ligated into the plasmid as long as they have the complementary ‘sticky ends’. The ligation is performed by the enzyme DNA ligase.

Genomic Library – a library of an organism’s DNA can be digested with a restriction endonuclease and then ligated into a plasmid. Each plasmid then has a different part of an organism’s genes, but the whole genome is represented somewhere in the group of plasmids.

Reverse Transcriptase – the enzyme that is carried in retroviruses (like HIV) reverse transcribes RNA into DNA. That function is useful in research because mRNA from eukaryotic cells that has already been modified, can then be reverse transcribed using that enzyme to convert mRNA back into DNA (called cDNA). This is useful because one can obtain the DNA coding sequence for a eukaryotic gene without all the intervening non-coding sequences, introns.

Gel electrophoresis – DNA is digested by a restriction endonuclease and the pieces of DNA can then be separated based on their size by using a gel matrix – so small sections of DNA move through the matrix faster than larger pieces. The actual separation through the gel is accomplishe by applying an electrical current through the gel. DNA, which has a negative charge, moves through the gel towards the positive pole.

Hybridization/Probes – gel electrophoresis is the basis of other types of techniques that lead to identifying certain genes. In a Southern hybridization, digested genomic DNA is separated by electrophoresis and the DNA is then transferred to a membrane. The membrane is then hybridized with a ‘labeled’ segment of the gene understudy called a probe (the label may be radioactive or other chemical labeling). When the hybridized filter is exposed to x-ray film, only the band with labeled probe hybridized to it will be shown on the film.

RFLP – restriction fragment length polymorphism. This is a widely used process to compare closely related genes for slight sequence differences. It is based on having restriction sites at different places in the genes with sequence differences. This causes one gene to have different bands which result from a gel electrophoresis than a known sample. This is used in forensics, medicine, and many other areas of research.

Polymerase chain reaction – PCR – This technique uses DNA polymerase that was isolated from bacteria which grow in thermal springs in Yellowstone Park. The enzyme is heat stable even at temperatures close to boiling. It functions as a normal DNA polymerase and is used to generate multiple copies of a stretch of DNA through a series of cycles in which the DNA is denatured, annealed to a primer, then elongated with the polymerase. PCR has revolutionized DNA research because large quantities of DNA can be made from a single copy of a gene.