Genetics Exam 4 Study Guide Chapter 17 Lectures 29 30 Additional Notes Chapter Concepts o Recombinant DNA technology creates combinations of DNA sequences from different sources o A common application of recombinant DNA technology is to clone a DNA segment of interest o For some cloning applications specific DNA segments are inserted into vectors to create recombinant DNA molecules that are transferred into eukaryotic or prokaryotic host cells such as bacteria where the recombinant DNA replicates as the host cells divide o DNA libraries are collections of cloned DNA o DNA segments can be amplified quickly and cloned millions of times using the polymerase chain reaction PCR o DNA RNA and proteins can be analyzed using a range of molecular techniques o DNA sequencing reveals the nucleotide composition of DNA and major developments in sequencing technologies have advanced many areas of modern genetics research particularly genomics o Recombinant DNA technology has revolutionized our ability to investigate the genomes of diverse species and led to the modern revolution in genomics Learning Objectives Be able to define the term recombinant DNA o Recombinant DNA from different sources palindromic DNA sequence is Know what restriction enzymes do what organisms have them and what a DNA created by joining together pieces of DNA o A restriction enzyme recognizes and binds to DNA at a specific nucleotide sequence restriction site then it cuts both strands of DNA within the sequence by cleaving the phosphodiester backbone of DNA Useful for cloning due to their ability to accurately and reproducibly cut genomic DNA into fragments at specific restriction sites Restriction enzymes are produced by bacteria as a defense mechanism against infection by viruses They restrict or prevent viral infection by degrading the DNA of invading viruses o Palindromic DNA sequence a double stranded DNA segment where each strand s base sequence is identical when read 5 to 3 Exhibited by most restriction sites Be able to do restriction enzyme mapping problems like the ones in the Be able to describe how restriction enzymes are used in cloning DNA homework assignment o Steps in Cloning a Gene Obtain isolated DNA from the organism Cut the DNA with restriction enzymes to isolate the desired fragment s Insert the DNA fragment s into a cloning vector Introduce recombinant DNA into a host cell such as E coli Grow the host and re isolate recombinant DNA clones o To distinguish host cells that have taken up vectors from host cells that have not the vector should carry a selectable marker gene usually an antibiotic resistance gene or the gene for an enzyme absent from the host cell also called blue white selection For lac Z insertion of recombinant DNA will disrupt the lac Z gene and functional B galactosidase will not be produced therefore if B galactosidase is present no insertion the plasmid will be blue and if it is not present proper insertion it will be white Know what a cloning vector is and why different vectors plasmid lambda and BAC vectors are used in different situations o Cloning vector DNA molecule that accepts DNA fragments and replicates inserted DNA fragments when placed into host cells Contains several restriction sites that allow insertion of the DNA fragments to be cloned o Plasmid an extrachromosomal circular DNA molecule that replicates independently of the host chromosome Plasmids are introduced into bacteria via transformation Only accepts inserts up to 25 kb o Lambda phage vector that can carry inserts up to 45 kb more than twice as long as DNA inserts in most plasmid vectors o Bacterial Artifical Chromosomes BACs essentially very large vectors that have low copy number plasmids that can accept DNA inserts in the 100 to 300 kb range F plasmid o Several types of vectors are used because they differ in the size of the inserts they can carry Know the difference between a genomic library and a cDNA library what each of these libraries are used for o DNA Library o Genomic Library A collection of cloned DNA samples includes the genomic library and the cDNA library A collection of clones that contains all the DNA sequences of an organism s genome isolated from chromosomes including the coding and noncoding sequences Used for cloning regulatory sequences promoters studying genome organization and sequencing of whole genomes o cDNA Library from mRNA isolated from cells or tissues A collection of clones that contains DNA copies cDNA Unlike a genomic library a cDNA library only contains expressed genes coding sequences exons and therefore is useful for identifying and studying genes expressed in certain cells or tissues under certain conditions Can be used to compare expressed genes from normal tissue and diseased tissue Be able to explain how double stranded cDNA is made from mRNA o mRNA oligo dT primer reverse transcriptase single stranded cDNA RNase DNA polymerase I DNA ligase double stranded cDNA Understand how you can screen a library to identify clones carrying a specific gene by use of DNA hybridization o Bacterial clones from the library are grown on nutrient agar plates where they form hundreds to thousands of colonies Colonies of the library are replicated by overlaying the plate with a DNA binding membrane such as nylon o Colonies are transferred to the membrane lysed and DNA is denatured o Membrane is placed in a heat sealed bag with a solution containing the labeled probe the probe then hybridizes with denatured DNA from colonies o Membrane is rinsed to remove excess probe and then dried X ray film is placed over the filter for autoradiography o Using the original plate cells are picked from the colony that hybridized to the probe o Cells are transferred to a medium for growth and further analysis Know what a Southern blot is and what a Northern blot is be able to outline how they are done and understand what they are used for Detection of DNA sequences on a gel used to study o Southern Blot gene structure 2 components Process Separation of DNA fragments by gel electrophoresis used to characterize the number of fragments produced by restriction digestion and to estimate their MWs Hybridization of the fragments using labeled probes DNA samples cut with restriction enzymes are loaded on a agarose gel for electrophoresis DNA is separated by gel electrophoresis but invisible to the naked eye DNA binding filter paper towels and a weight are placed on the gel Buffer passes upward by capillary action
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