Chapter on Recombinant DNA De able to define the term recombinant DNA Recombinant DNA is DNA from two different sources spliced together To create a recombinant DNA molecule DNA from a host cell is isolated and cut with restriction enzymes enzymes that cut DNA at specific nucleotide sequences complementary to specific restriction enzymes and then added to a solution containing carrier or vector DNA fragments fragments able to enter the host cell cut with the same restriction enzymes DNA ligase is added to the solution and the fragments anneal creating recombinant DNA fragments that can now enter the host cell and be replicated DNA from host Vector DNA Recombinant DNA molecule Know what restriction enzymes do what organisms have them and what a palindromic DNA sequence is Restriction enzymes are proteins produced by bacteria as viral defense mechanisms They cut DNA at recognition sequences nucleotide sequences specific to that restriction enzyme These recognition sequences are referred to as palindromic meaning the sequences compliment is the same as the original read backwards they read the same front to back The sequence GAATTC is considered palindromic because its compliment sequence CTTAAG is the same sequence read backwards Be able to describe how restriction enzymes are used in cloning DNA DNA cloning is the use of recombinant DNA molecules to trick a host cell into replicating the same target DNA sequence over and over again Restriction enzymes of known effects are used to isolate vector sequences sequences of DNA known as plasmids that are able to enter the host cell and replicate independently of the chromosome the same restriction enzyme is used to create complimentary sequences from the outside target DNA to be inserted and cloned Once these complimentary fragments are joined by DNA ligase they are considered recombinant vectors and are examples of recombinant DNA molecules These molecules can now enter the host cell and use it to replicate clone the added in sequence Without specific restriction enzymes there would be no way to create complimentary sequences able to be joined together Know the difference between a genomic library and a cDNA library what each of these libraries are used for Genomic libraries are cloned fragments of DNA isolated directly from a chromosome unedited DNA This means that it will contain all repetitive and regulatory sequences and introns that are processed out before translation The creation of genomic libraries are used to study regulatory DNA sequences such as promoters and suppressers the organization of an organisms genome and for sequencing of an entire genome like the human genome project cDNA libraries are cloned DNA sequences from mRNA fragments using reverse transcriptase creates a DNA molecule from a transcript rather than a transcript being created from a DNA molecule This creates much more specific and smaller libraries used for studying specific expressed genes Know the steps required to convert mRNA into double stranded cDNA made from mRNA Understand how you can screen a library to identify clones carrying a specific gene by use of DNA hybridization To screen a library of cloned DNA such as cDNA the already cloned DNA from a host cell must be isolated and denatured through heating Then a radioactively labeled probe sequence of DNA complimentary to the sequence being screened for in the cloned DNA radioactivity allows the probe to be found using X rays is denatured and cooled rapidly to ensure it remains single stranded This single stranded probe is then added to the solution containing the still denatured clone DNA The solution is allowed to cool and thus the single stranded probe should bind to its compliment sequence on the clone DNA The newly formed DNA strand can be viewed under X ray to see if the probe bonded to it If so then the desired gene is present in the clone DNA For example if you were screening a DNA library for gene A you would add a probe that compliments the sequence of gene A If the clone DNA contains the sequence for gene A the probe will bind to it and the molecule will now contain a radioactive tag for gene A 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 Southern blotting is the use of gel electrophoresis to detect specific DNA sequences and use them to study the structure of the genes The same principle of hybridization is used For example if you want to test to see if gene R is located on the area of DNA cut by restriction enzyme A or restriction enzyme B you would use Southern blotting Southern blots are created by cutting DNA with one or more restriction enzymes such as A and B and then separating them using gel electrophoresis Radioactive size markers are run through the first lane DNA cut with restriction enzyme A is loaded into the second lane and DNA cut with restriction enzyme B is loaded into the third lane All the DNA is then denatured and transferred onto a piece of filter paper that the single stranded DNA molecules can bind to This filter is placed in a solution with single stranded radioactive probes for gene R and the molecules are allowed to renature When viewed under X ray all the size markers in lane 1 are visible and in lane 2 and 3 only the strands that hybridized with the probe are visible If visible under X ray then you know that the area of the DNA that is cut by restriction enzyme A or B contains gene R Northern blotting tests for the presence of mRNA fragments complimentary to a cloned gene It is used to determine if a specific gene is being actively expressed in a given cell This is done by running the mRNA isolated from the target cell through a gel The RNA blots are then transferred to a filter paper as in southern blotting and exposed to radioactively labeled single stranded cloned DNA fragments of the gene sequence being searched for Complementary molecules will hybridize and show up under X ray If the gene is being expressed in the cell its mRNA will hybridize with the clone gene sequence Understand what PCR is used for and how it is done and know what the important considerations are in getting PCR to amplify a specific DNA sequence length of the primers and choice of the annealing temperature Understand why a thermostable DNA polymerase is important in PCR and which organisms make thermostable DNA polymerases PCR stands for polymerase chain reaction It is the cloning and amplification
View Full Document
Unlocking...