BIOL 4610 1st Edition Lecture 3 Outline of Current Lecture I. Genetic analysisII. SelectionIII. Manipulating DNA/RNAIV. Inactivating specific genesCurrent LectureChapter 5Classical genetics: looking for observable differences Look at DNA and find the gene responsible for one set to be green and the other yellow Take gene and use it to make proteins Reverse genetics: don’t need to see observable phenotype, but can go into database and identify protein coding sequence to manipulate it to make an observable difference in the organism5.1. Genetic analysis of mutations to identify and study genes Vocabulary Mutant/mutation: change in very small part of DNA sequence Allele- mutation that is naturally occurring Genotype: set of alleles (one from mom and one from dad) that we are looking at Same alleles= homozygous, exactly identical same set of alleles Allele A, allele B- heterozygotes, two different alleles of the same gene Phenotype: physical traits that we attribute back to our genotypeThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.Inheriting mutations – recessive usually a loss of function yellow comes from mom and blue from dad. to inherent recessive alleles, must have both alleles being mutant Restriction enzymes (endonucleases) cut DNA into small fragments and produce ‘sticky ends’. Use enzymes “endonucleases” that cut nucleic acids (DNA) at specific sequences (usually palindromes) Palindromes read the same way backwards and forwards Cuts at palindrome sequences no matter where they are Produces sticky ends that are single strands Plasmid vectors – used for cloning isolated DNA fragments Small circular DNA pieces of bacteria Independent of chromosome but also replicates during cell division Origin of replication Select a drug resistance gene (ampr- ampicillin resistance) Polylinker- has sites for multiple restriction enzymes to cut at Inserting fragments into vectors Cut plasmid and DNA of interest with same restriction enzyme Insert DNA into plasmid vector using DNA ligase Transformation: bacteria takes up the recombinant DNA plasmid Increase this by punching “holes” into the bacteria cell wall Not all bacteria takes up the plasmid, but still happens enough that we can use this Selection: making use of drug resistance gene (ampicillin resistance gene) Cells that don’t take up plasmid will dieGel electrophoresis: use same restriction enzyme to cut gene back out Load insulin gene and vector into gel• Nucleic acid is negatively chargedDNA library Multiple nucleic acid pieces (gene) Use restriction enzymes, may use many different ones for the whole genome Can create libraries- what mRNA is expressed in particular tissues or at particular times. RNA is too unstable to study transcript Have my isolated nucleic acid fragments – now what? Manipulating DNA/RNA 1. Southern blotting – detect specific DNA sequences2. Northern blotting – RNA Run the RNA on a gel and don’t know where our gene of interest is actually located In picture, we can see that it’s absent in the first column, a little bit in middle, and very abundant in far right column 3. Polymerase chain reaction (PCR): directly amplify region of DNA Uses Thermus aquaticus (Taq) polymerase- specialized DNA polymerase that works at really high temperatures Increase the temp (98 degrees Celsius) to separate DNA into single strands Design primers that are complimentary to the exact gene that we want Cool slightly (50-60 degrees C) to allow primers to find where they compliment5.5. Inactivating the function of specific genes in eukaryotes Reasonably easy to find new genes based upon sequencing of genomes, cDNA libraries, etc. But, knowing what these ‘new’ proteins actually do often times is unclear.Can try to disrupt the gene and see what types of mutant phenotypes develop. Replace normal gene with other sequences in eukaryotes Produce an altered gene and then introduce it into the germ line via homologous recombination. This is called a knockout animal using embryonic stem cells (ES