Chapter 17 Be able to define the term recombinant DNA Recombinant DNA is DNA created by joining together pieces of DNA from The method used to clone DNA is called recombinant DNA technology and is different sources often known as gene splicing Know what restriction enzymes do what organisms have them and what a palindromic DNA sequence is 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 A restriction enzyme recognizes and binds to DNA at a specific nucleotide sequence called a restriction site The enzyme then cuts both strands of the DNA within that sequence by cleaving the phosphodiester backbone of DNA A palindromic DNA sequence is a double stranded DNA segment where each strand s base sequence is identical when read 5 to 3 o For example 5 GAATTC 3 3 CTTAAG 5 Be able to do restriction enzyme mapping problems like the ones in the homework assignment Be able to describe how restriction enzymes are used in cloning DNA Restriction enzymes cut different types of DNA sequences The enzymes must cut complementary tails in both the DNA from the organism and the DNA from the vector in order for cloning to occur Know what a cloning vector is and why different vectors plasmid lambda and BAC vectors are used in different situations Cloning vectors are DNA molecules that accept DNA fragments and replicate inserted DNA fragments when placed into host cells Vector contains several restriction sites that allow insertion of DNA Vectors differ in terms of the host cells they are able to enter and in the size fragments to be cloned of inserts they can carry o Plasmid vectors can only accept inserted pieces of DNA about 25 kb in size on slides 10 kbp o Lambda can accept up to 45 kb on slides 10 50 kbp o BAC vectors can accept up to 100 300 kb but have low copy number on slides 100 kbp Know the difference between a genomic library and a cDNA library what each of these libraries are used for A genomic library is a collection of clones that contains all the DNA sequences of an organism s genome o Cloned fragments of DNA isolated from chromosomes o Used for cloning regulatory sequences promoters studying genome organization sequencing of whole genomes cDNA libraries contains DNA copies called cDNA which are made from the mRNA molecules of a cell population and therefore represent the genes being expressed in the cells at the time the library was made o Cloned DNA copies of mRNA isolated from cells or tissues o Used for isolating expressed genes Be able to explain how mRNA is separated from bulk RNA in the cell and how double stranded cDNA is made from mRNA mRNA from a population of cells of interest are isolated typically cells that express an abundance of mRNA for the genes to be cloned mRNAs are mixed with oligo dT primers short single stranded sequences of T nucleotides that anneal to the poly A tail the enzyme reverse transcriptase extends oligo dT primer and synthesizes a complementary DNA copy of the mRNA sequence creating mRNA DNA double stranded hybrid molecule The RNA in the hybrid molecule can be enzymatically digested or chemically degraded and another opposing strand of DNA is synthesized by DNA polymerase DNA polymerase I is added and then DNA ligase is added to seal gaps Understand how you can screen a library to identify clones carrying a specific gene by use of DNA hybridization To screen library with probe o Bacterial clones from library are grown on nutrient plates where they form colonies o A replica of the colonies on each plate is made by pressing nylon or nitrocellulose membrane onto plates surface which transfers pattern of bacterial colonies to membrane o Colonies on membrane are lysed and DNA is denatured o DNA on membrane is incubated with labeled nucleic acid probe First probe is heated then cooled to form single stranded molecules then it is added to a solution containing the membrane o If nucleotide sequence of DNA on membrane is complementary to probe a double stranded DNA DNA hybrid molecule is formed and this step is called hybridization o Unbound probe molecules washed away and membrane is assayed to detect hybrid molecules that remain o If radioactive probe used film is placed on membrane and spots will form on the film Spots represent colonies on the plate containing cloned gene of interest o Position of spots on film used to identify corresponding colonies on the plate 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 blot method can be used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments o Also can be used to identify fragments carrying specific genes in genomic DNA digested with a restriction enzyme o It can also help identify the number of copies of a particular sequence or gene that are present in a genome o Steps for southern blotting DNA samples cut with restriction enzymes are loaded on agarose gel for electrophoresis DNA at this point is separated by electrophoresis but invisible to the naked eye DNA is then denatured to form single stranded fragments and gel is placed on sponge wick DNA binding filter paper towels and weight are placed on gel Buffer passes upward by capillary action transferring DNA fragments to filter Filter placed in heat sealed food bag with solution containing labeled probe Filter is washed to remove unbound probe dried and film is applied for autoradiography Northern blot method probes for the presence of mRNA complementary to a cloned gene o They provide info about the expression of specific genes and are used to study patterns of gene expression o They also detect alternatively spliced mRNA and are used to derived other information about transcribed mRNAs o Can also be used to measure size of genes mRNA transcripts o Steps for northern blotting mRNA is extracted from specific cell or tissue separated by gel electrophoresis and RNA is transferred to a membrane Membrane is exposed to a labeled probe If mRNA complementary to DNA probe is present complementary sequences will hybridize and be detected as a band on the film Understand what PCR is used for and how it is done know the function is of each of the steps in the PCR cycle denaturation annealing extension why a thermostable DNA polymerase is important in PCR and which organisms make thermostable DNA
View Full Document
Unlocking...