Chapter 4. The Genomic Biologist’s Toolkit Contents 4.7. Genome Annotation 4. Genomic Biologists tool kit 4.7.1. Using Bioinformatic Tools to Identify Putative 4.1. Restriction Endonucleases – making “sticky ends” Protein Coding Genes 4.7.2. Comparison of predicted sequences with known 4.2. Cloning Vectors sequences (at NCBI) 4.2.1. Simple Cloning Vectors 4.7.3. Published Genomes 4.2.2. Expression Vectors 4.2.3. Shuttle Vectors 4.2.4. Phage Vectors 4.2.5. Artificial Chromosome Vectors 4.3. Methods for Sequence Amplification 4.3.1. Polymerase Chain Reaction 4.3.2. Cloning Recombinant DNA 4.3.3. Cloning DNA in Expression Vectors 4.3.4. Making Complementary DNA (cDNA) 4.3.5. Cloning a cDNA Library 4.4. Genomic Libraries 4.4.1. Cloning in YAC Vectors 4.4.2. Cloning in BAC Vectors 4.5. DNA sequencing 4.5.1. Electrophoresis 4.5.2. Sanger Dideoxy Sequencing 4.5.3. Capillary Sequencers 4.5.4. Next Generation Sequencing 4.5.5. 3rd Generation Sequencing 4.6. DNA Sequencing Strategies 4.6.1. Map-based Strategies 4.6.2. Whole Genome Shotgun Sequencing CONCEPTS OF GENOMIC BIOLOGY Page 4-1 4.1. RESTRICTION ENDONUCLEASES (RETURN) CHAPTER 4. THE GENOMIC BIOLOGIST’S Restriction endonucleases (restriction enzymes) each TOOLKIT (RETURN) recognize a specific DNA sequence (restriction site), and break a phosphodiester linkage between a 3’ carbon and phosphate within that sequence. Restriction enzymes are used to create DNA fragments for cloning and to Genomic Biology has 3 important branches, i.e. analyze positions of restriction sites in cloned or Structural Genomics, Comparative genomics, and genomic DNA. A specific restriction enzyme digests cut Functional genomics. The ultimate goal of these DNA at the same sites in every molecule if allowed to branches is, respectively; the sequencing of genes and cut to completion. Thus, this is a method whereby all genomes; the comparison of these sequenced genes copies of genomes or any other longer sequence can be and genomes, and an understanding of how genes and reproducibly cut into identical fragments. genomes work to produce the complex phenotypes of The first three letters of the name of a restriction all organisms. enzyme are derived from the genus and species of the A set of molecular genetic technologies was/is critical organism from which it was isolated. Additional letters to our ability to pursue the goals described above. The often denote the bacterial strain from which the Genomic Biologists Tool Kit is provides a brief restriction enzyme was isolated, and if multiple enzymes understanding of these critical tools, and how they are are isolated from the same strain, they are given Roman used in the investigation of genomes. While the numerals. For example, the restriction enzyme EcoRI, is techniques are intrinsically laboratory tools, the nature the first enzyme isolated from the RY13-strain of of what they can do and how they work can be readily Escherichia coli. studied using bioinformatic resources. Bacteria produce restriction endonucleases to defend against bacteriophages (viruses), and each restriction CONCEPTS OF GENOMIC BIOLOGY Page 4-2 Table 4.1. Characteristics of Some Restriction Enzymes CONCEPTS OF GENOMIC BIOLOGY Page 4-3 enzyme recognizes a completely unique DNA sequence from degrading host cell DNA, while invading bacter- where it cuts the DNA strands (see Table 4.1 & Figure iophage DNA is unmethylated and readily degraded. 4.1). The specific restriction enzyme recognition sites in Many restriction sites are sequences of 4, 6, or 8 the bacterial DNA are often limited in the genome of the base pairs in length and have identical sequences from organism from which it comes, but they are abundant in 5’ to 3’ on each strand. These sequences are referred to the genome of the bacteriophage. Also the DNA of the as palindromic DNA sequences. Other restriction sites host cell can be modified by methylation, which are not completely symmetrical and/or differ in length prevents the restriction enzymes of the host cell from 4, 6, or 8 nucleotide pairs (Table 4.1 & Figure 4.1). As shown in the figure on the left, the nature of the fragment ends produced when a restriction enzyme produces DNA fragments can vary. Some enzymes produce fragments where the two strands are equal in length. This is referred to as blunt ends. Other enzymes produce fragments where the two strands are unequal in length. These are referred to as either 5’ sticky ends, or 3’ sticky ends. Overhanging sticky ends provide a basis for combining DNA fragments produced by the same restriction enzyme from different DNA sources. This process was the original method used to produce recombinant DNA molecules. The application of restriction endonucleases to the cloning of DNA is further discussed in DNA Cloning video that can be viewed by clicking on the link. Note that part of this video will be discussed in detail in the next Figure 4.1. Restriction site sequences and section of the Genomic Biologist’s Toolkit, but the first cut locations of: a) SmaI; b) BamHI, and c) part of the video is a good demonstration of how PstI. CONCEPTS OF GENOMIC BIOLOGY Page 4-4 restriction enzymes work and how they can be used to Note that we have previously discussed SNPs as a create recombinant DNA molecules for cloning DNA. type of Sequence Tagged Site (STS). As single nucleotide changes in the genome sequence, consider the effect of an SNP that happens to occur in a restriction endonuclease recognition site. The result would be the loss of a restriction site at that SNP. This site would no longer be cut by the enzyme, and thus new fragments having different sizes would be produced. This is called Restriction Fragment Length Polymorphism (RFLP). Thus, and RFLP is an SNP that happens to occur in a restriction site in the DNA. A famous RFLP is associated with Sickle Cell Disease, and is further described in the accompanying video. Figure 4.2. Using restriction enzyme, EcoRI to make recombin-ant 4.2. CLONING VECTORS (RETURN) DNA. The procedure relies on the 3’-overhanging “sticky ends”. The process of “DNA cloning” involves a set of An additional application of restriction enzymes experimental methods in molecular biology that are involves the production of a res-triction map. A used to assemble recombinant DNA molecules and to restriction map is shows the relative position of direct their replication within host organisms. The use restriction sites for multiple restriction enzymes in a of the word cloning refers to the fact that the method piece of linear or circular DNA. Prior to the availability involves the replication of one molecule to produce a of genomic sequences, restriction mapping was an population of cells with identical DNA molecules. important tool used to characterize cloned DNA Molecular cloning generally uses DNA sequences from fragments. The production of a restriction map for a two different organisms: 1) the organism that is the circular DNA is shown in the Restriction Mapping video. source of the DNA to be cloned, and 2) the organism that will serve as the living host for replication of the CONCEPTS OF GENOMIC BIOLOGY Page 4-5 recombinant DNA. Molecular cloning methods are antibiotic or that permits cells to make an amino acid central to many areas of biology, biotechnology, and required for growth. medicine, including DNA sequencing. These are the basic requirements that all modern The DNA from host organism in a cloning cloning vectors contain, but beyond these basic experiment, often called a vector, typically has 3 things: requirements, there can be a number of additional features that make specific vectors useful for various 1) Sequences necessary to produce recombinant DNA purposes. Thus, several types of cloning vectors have and facilitate entry into the host organism. Typically, been constructed, each with different molecular this can be one or more “unique” restriction sites. properties and cloning capacities. “Unique” in this context means that these are restriction sites will permit cutting the vector at only 4.2.1. Simple Cloning Vectors (RETURN) one location. Most vectors contain unique restriction The most common vectors are used to clone sites for a number of different restriction enzymes. recombinant DNA in bacterial cells, typically E. coli. This is called a polylinker or multiple cloning site, and Simple cloning vectors are constructed from plasmids can make the use of the vector much easier. common in many bacterial cells. In fact plasmids are 2) An origin of replication for the host organism to circles of dsDNA (double stranded) much smaller than facilitate replication of the recombinant DNA in the the bacterial chromosome that include replication host cell. Typically this sequence controls the origins (ori sequence) needed for replication in bacterial number of copies of the vector that can be made in cells that naturally carry DNA between different one cell. bacteria. An example of a typical E. coli cloning vector is 3) In order to facilitate identification of cells that contain pUC19 (2,686bp). The more modern version of pUC19 is the vector containing recombinant DNA, a gene that pBluescript II. The features of this plasmid are shown in can be expressed in the host and that provides a Figrue 4.2. “selectable” marker for the presence of recombinant More information about cloning DNA in plasmid DNA is provided. Often the selectable marker gene vectors can be found in Molecular Cell Biology, 4th will be a gene that makes cells resistant to a specific edition, Section 7.1. This can be downloaded from NCBI by clicking on the link. The use of simple cloning vectors CONCEPTS OF GENOMIC BIOLOGY Page 4-6 Figure 4.3.