GEN 3022 1st Edition Lecture 32 Outline of Last Lecture I Gene conversion a Example I b Gap repair synthesis vs DNA mismatch repair II Transposition a Cut and paste mechanism b Reverse transcriptase III Homologous Recombination a Holliday model b Recombinant and nonrecombinant chromosomes c Heteroduplex DNA Outline of Current Lecture I Recombinant DNA technology a Discovery b Importance of recombinant DNA technology II Gene cloning a Cloning experiments i cDNA ii vector DNA iii preparation of DNA iv host cell b restriction enzymes III Steps in gene cloning of human B globin gene a Plasmid AmpR and lacZ b cDNA c Restriction enzymes d Recombinant event e Testing for the desired gene f Net Result of cloning IV Polymerase Chain Reaction a Starting materials i Template DNA ii Oligonucleotide primers These 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 iii Deoxynucleoside triphosphates iv Taq polymerase b Process c Result d Reverse transcriptase PCR Current Lecture I Recombinant DNA technology the use of in vitro molecular techniques to isolate and manipulate fragments of DNA a Discovery discovered in early 1970s by researchers at Stanford who were trying to construct chimeric molecules called recombinant DNA molecules This led to introducing the molecules into living cells where they are replicated to produce many copies b Importance of recombinant DNA technology Recombinant DNA technology opened the door to gene cloning research both of which have been critical to understanding gene structure and function II Gene cloning the technique of isolating and making many copies of a gene a Cloning experiments involve two kinds of molecules cDNA or chromosomal DNA and vector DNA i Preparation of chromosomal DNA or cDNA cellular tissue is obtained from organism of interest the cells are lysed broken open and the DNA is extracted and purified ii Host cell cell that harbors the vector When a vector is replicated inside a host cell the DNA that it carries is also replicated b cDNA also known as complementary DNA c Vectors serves as the carrier for the DNA segment that is to be cloned Can replicate independently of the host chromosomal DNA These vectors were originally derived from plasmids and viruses d Restriction enzymes also called restriction endonucleases Bind to specific sequences usually palindromic sequences in the DNA and cleave strands at two defined locations one on each strand to create sticky ends i The ends will hydrogen bond to each other due to their complementary sequences Some generate blunt ends though ii Made naturally by many types of bacteria protect bacterial cells from invasion by foreign DNA especially bacteriophages III Steps in gene cloning of human B globin gene a Plasmid AmpR and lacZ AmpR confers antibiotic resistance to the host cell Therefore only cells that have taken up the vector will grow on an ampicillin IV plate LacZ encodes for B galactosidase and provides a means to determine which cells grew on the ampicillin plate and contain the cloned gene This is called screening b cDNA contains desired gene Combined with vector DNA and restriction enzymes Formed using reverse transcriptase and can be single or double stranded Important advantage is the lack of introns which allows researchers to focus their attention on the coding sequence of a gene and the expression of the encoded protein c Restriction enzymes cut up cDNA and plasmid DNA vector at specific sites d Recombinant event the plasmid DNA and cDNA combine to form recombinant vectors Only some of these contain the desired gene e Testing for the desired gene the vectors are inserted into host cells where they replicate This process is called transformation The cells are then allowed to grow on an ampicillin plate Cells that contain functional B galactosidase do not contain the desired gene and will turn blue The cells that remain white contain the desired recombinant vector f Net Result of cloning goal is to produce an enormous number of copies of a gene Even though a single bacterial cell takes up just one copy of a vector during transformation the cloned gene is amplified when the vector is replicated in the host cell or when the bacterial cell itself divides about every 20 minutes Polymerase Chain Reaction a technique developed by Kary Mullis in 1985 to copy DNA without the aid of vectors and host cells Can also be used to amplify chromosomal DNA not just cDNA but it is not as specific This is what is used to amplify DNA samples found at crime scenes a Starting materials i Template DNA contains the region that is amplified by PCR ii Oligonucleotide primers complementary to sequences at the ends of the DNA fragment to be amplified Synthetic and usually 15 20 nucleotides in length iii Deoxynucleoside triphosphates provide the precursors for DNA synthesis iv Taq polymerase DNA polymerase that is isolated from the bacterium Thermusaquaticus Necessary because it is stable under high temperatures and PCR involves heating steps that would inactivate many other DNA polymerases b Process carried out in a thermocycler that is programmed to maintain a certain range of temperatures and automates the timing of each cycle Primers are selected that bind to either side of the desired gene Many PCR cycles occur and each cycle produces copies of the region of interest following the general pattern of denaturing annealing synthesis c Result a typical PCR is 20 30 cycles which increases the DNA sequence to 2 20 fold of the original assuming 100 efficiency d Reverse transcriptase PCR used to clone and quantitate RNA in living cells RNA is first isolated from the sample and mixed with reverse transcriptase and a primer that will anneal to the 3 end of the RNA of interest This generates a single stranded cDNA which can be used as a template DNA in conventional PCR RT PCR is very sensitive and can detect the expression of small amounts of RNA in a single cell