Objectives for Exam 4 Genetics Fall 2014 Chapter on Recombinant DNA Chapter 17 De able to define the term recombinant DNA DNA created by joining together pieces of DNA from different sources o Recombinant DNA technology is used to clone DNA Know what restriction enzymes do what organisms have them and what a palindromic DNA sequence is DNA cutting enzymes o They recognize and bind to DNA at a specific nucleotide sequence called a restriction site The enzyme then cuts both strands of DNA within the sequence by cleaving the phosphodiester backbone of DNA o They are useful in cloning because they can cut genomic DNA into fragments They are present in bacteria o Restriction sites have a form of symmetry known as a palindrome o Palindrome DNA sequence a double stranded DNA segment where each strand s base sequence is identical when read 5 to 3 for example 5 GAATTC 3 3 CTTAAG 5 The restriction enzyme recognizes it s particular restriction site and cuts the DNA in a characteristic DNA pattern o o o Be able to do restriction enzyme mapping problems like the ones in the homework assignment QUESTION 5 MG ASSIGNMENT 7 Be able to describe how restriction enzymes are used in cloning DNA Know what a cloning vector is and why different vectors plasmid lambda and BAC vectors are used in different situations Vector DNA molecules that accept DNA fragments and replicate inserted DNA fragments when placed into host cells Plasmids are still workhorses for many applications of recombinant DNA technology but they have a limitation because they are small they can only accept inserted pieces of DNA up to about 25 kb in size and most plasmids can often only accept substantially smaller pieces Therefore as recombinant DNA technology has developed and it has become desirable to clone large pieces of DNA other vectors have been developed primarily for their ability to accept larger pieces of DNA and because they can be used with other types of host cells beside bacteria Know the difference between a genomic library and a cDNA library and what each of these libraries are used for Genomic library consists of many overlapping fragments of the genome which at least one copy of every DNA sequence in an organisms genome which in summary span the entire genome AKA a collection of clones that contains all the DNA sequences of an organism s genome o Used for cloning regulatory sequences promoters studying genome organization sequencing of whole genomes cDNA library contain DNA copies cDNA which are made from mRNA molecules of a cell population and therefore represent the genes being expressed in the cells at the time the library was made It contains only EXPRESSED genes o Used to identify and study genes expressed in certain cells or tissues under certain conditions They provide and snapshot of the genes that were transcriptionally active in a tissue at a particular time because the relative amount of cDNA in a library is equivalent to the amount of starting mRNA isolated from the tissue and used to make the library Be able to explain how mRNA is separated from bulk RNA in the cell and how double stranded cDNA is made from mRNA mRNA is separated from bulk RNA in the cell a cDNA library is prepared by isolating mRNA from a population of cells of interest typically cells that express an abundance of mRNA from the genes to be cloned The next step is to mix mRNAs with oligo dT primers short single stranded sequences of the T nucleotide that anneal to the poly A tail Double stranded cDNA is made from mRNA reverse transcriptase extends the oligo dT primer and synthesizes a complementary DNA copy of the mRNA sequence creating an 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 Alternatively cDNA can also be synthesized using primers that bind randomly to mRNA and the first strand to cDNA to eventually create double stranded cDNA from the original mRNA strand Understand how you can screen a library to identify clones carrying a specific gene by use of DNA hybridization You can screen the library with a probe any DNA RNA sequence that s complementary to some part of a cloned sequence present in the library 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 How southern blotting is done DNA is cut into fragments with one or more restriction enzymes and the fragments are separated by gel electrophoresis In preparation for hybridization the DNA in the gel is denatured with alkaline treatment to form single stranded fragments The gel is the overlaid with a DNA binding memebrane usually nylon Transfer of the DNA fragments to the membrane is accomplished by placing the membrane and gel on a wick sitting in a buffer solution Layers of paper towles or blotting papers are placed on top of the membrane and held in place with a weight Capillary action draws buffer up through the gel transferring the DNA fragments from the gel to the membrane The membrane is placed in a heat sealed bag with a labeled single stranded DNA probe for hybridization DNA fragments on the probe that are complementary to the probe s nucleotide sequence bind to the probe to form double stranded hybrids Excess probe is the washed away and the hybridized fragments are then seen on the film What southern blotting is used for can be used to identify which clones in a library contain a given DNA sequence and to characterize the size of the fragments They can also be used to identify fragments carrying specific genes in genomic DNA digested with a restriction enzyme How northern blotting is done mRNA is extracted from a specific cell or tissue type separated by gel electrophoresis and RNA is transferred to a membrane The membrane is the exposed to a labeled single stranded DNA probe derived from a cloned copy of the gene If mRNA complementary to the DNA probe is present the complementary sequences will hybridize and be detected as a band on the film What Northern blots are used for they provide information about the expression of specific genes and are used to study patterns of gene expression They can also characterize and quantify the transcriptional activity of genes in different samples 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
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