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Updated Summer 2015 Jerald D. Hendrix A. Recombinant DNA Technology 1. 2. 3. 4. 5. 6. 7. Restriction Endonucleases Creating a Recombinant DNA Library Properties of a Cloning Vector Screening a Recombinant DNA Library DNA Sequencing Polymerase Chain Reaction Bioinformatics Type II Restriction Endonuclease Recognizes a specific sequence (recognition sequence) on double stranded DNA, and . . . Cleaves the DNA molecule at the recognition site This makes Type II restriction endonucleases a very specific and precise molecular scissors to cut DNA. Recognition sequences are 4 – 8 nucleotide base pairs in length, with 6 bp sequences the most common Several hundred restriction endonucleases have been discovered Unless otherwise specified, the term “restriction endonuclease” implies “Type II” The term “restriction enzyme” is also used synonymously with “Type II restriction endonuclease” (and will be from this point on in the notes!) Type II Restriction Endonuclease (cont.) Restriction enzymes may make either staggered cuts or blunt cuts (flush cuts, straight cuts) In a blunt cut, the two phosphodiester bonds that are cut are directly across from each other, so each piece has double stranded DNA all the way to the end In a staggered cut, the two phosphodiester bonds that are cut are offset, so each piece has a short segment of single-stranded DNA at its end Type II Restriction Endonuclease (cont.) Most restriction sites are molecular palindromes (palindromic), meaning that the sequence on one strand reads the same as the sequence on the other strand, but in the opposite direction If the recognition site is palindromic and the enzyme makes a staggered cut, then the single stranded ends will be complementary to each other. These are called sticky ends. Sticky ends made with the same enzyme can hybridize, allowing DNA from more than one source to be spliced together. The segments are sealed together with a different enzyme, called DNA ligase. Type II Restriction Endonuclease (cont.) Example: EcoRI 5’ ↓ 3’ -N-N-G-A-A-T-T-C-N-N-N-N-C-T-T-A-A-G-N-N3’ ↑ 5’ 5’ 3’ -N-N-G A-A-T-T-C-N-N-N-N-C-T-T-A-A G-N-N3’ 5’ Definitions Recombinant DNA: A double stranded DNA molecule created by splicing DNA from different sources, using restriction enzymes and DNA ligase DNA cloning: using a bacterial species (most often, E. coli), to replicate recombinant DNA Vector: A small double stranded DNA molecule with an origin of replication for the bacterial host and a system for selecting recombinant DNA molecules of interest; most often an engineered bacterial plasmid or bacteriophage. Example: pUC18 Recombinant DNA genomic library: A collection of bacterial colonies with recombinant DNA (for example, an armadillo library), ideally containing the entire genome of the species Creating a Recombinant Library (Shotgun approach) Cut the vector DNA (pUC18) and the genomic DNA (armadillo DNA, if you want an armadillo library) with the same restriction enzyme (or combination of enzymes) Mix the vector and genomic DNA and ligate using DNA ligase After the ligation step, the mixture will basically contain three things: Genomic DNA without a vector Vector DNA (pUC18) without an insert (no genomic DNA) Recombinant DNA consisting of a vector molecule with a piece of genomic DNA inserted (spliced in) Creating a Recombinant Library (continued) Use the DNA mixture to transform competent E. coli cells. After transformation, there will basically be four kinds of E. coli in the tube: E. coli cells that were not transformed (didn’t get any new DNA) E. coli cells that were transformed with “Genomic only” E. coli cells that were transformed with “Vector only/No insert” E. coli cells that were transformed with “Recombinant plasmids/With Insert” Creating a Recombinant Library (continued) Plate the transformed E. coli onto X-gal (5-bromo-4chloro-3-indolyl-β-D-galactopyranoside) agar plates. These are selective and differential plates. X-gal agar contains ampicillin. If the E. coli cells weren’t transformed or got genomic DNA only (no vector/no plasmid), the ampicillin kills them. If the transformed E. coli got a vector only/no insert, it forms a blue colony. If the transformed E. coli got a vector with a genomic DNA insert, it forms a white colony. White colonies are further screened to determine which genes they contain. A total armadillo library might require screening of several thousand colonies! Origin of replication (ori) from one or more bacterial species “Shuttle vector” has origins from two or more species, allowing cloned genes to be shuttled from one species to another An antibiotic resistance gene (e.g. ampR). By using medium containing the antibiotic (ampicillin), only cells transformed with the vector DNA can survive. This selects against untransformed cells. One or more restriction enzyme recognition sites “Polylinker” – This is an engineered DNA segment containing recognition sites for several different enzymes, in tandem. A way to screen for vectors with inserts vs vectors without inserts Typically, this is a combination of the lac z gene (β-galactosidase gene) and the lac promoter sequence (required for transcription of the lac z gene). With no insert, a transformed cell will make βgalactosidase. The polylinker is engineered to overlap or sit between the lac p and lac z sequences. If there is an insert in the vector, it separates or disrupts lac p and lac z, so that β-galactosidase is not made. X-gal agar contains a synthetic substrate of β-galactosidase that turns blue if the enzyme is present (no insert). If there is an insert present, then β-galactosidase is not made, so the colonies are white. Two approaches Screen for expression of the heterologous protein Use a labelled DNA hybridization probe to search for colonies with homologous sequences, using blotting techniques Blotting techniques Southern Blotting: Use a labeled DNA probe to analyze DNA fragments, separated by agarose gel electrophoresis and transferred (“blotted”) onto nitrocellulose or nylon membrane sheets Blotting techniques Northern Blotting: Use a labeled DNA probe to analyze RNA molecules, separated by agarose gel electrophoresis and transferred (“blotted”) onto nitrocellulose or nylon membrane sheets Dot Blotting: The unknowns are simply spotted onto a membrane, then analyzed with a DNA probe Western Blotting: Not really a DNA technique. Analyzing proteins on a nylon membrane using a labeled antibody molecule as a probe Blotting techniques Northern Blotting: Use a labeled DNA probe to analyze RNA molecules, separated by agarose gel electrophoresis and transferred (“blotted”) onto nitrocellulose or nylon membrane sheets Dot Blotting: The unknowns are simply spotted onto a membrane, then analyzed with a DNA probe Western Blotting: Not really a DNA technique. Analyzing proteins on a nylon membrane using a labeled antibody molecule as a probe Approaches to obtaining a DNA probe Use a homologous sequence from a different (ideally one that is related) species Isolate mRNA for the gene of interest (for example, by using antibodies to immunoprecipitate the ribosome/nascent protein/mRNA complex). You then use reverse transcriptase to make a cDNA copy of the mRNA. Use DNA chemical synthesis techniques to create possible homologous sequences to the gene of interest, and test them as probes. After a segment of DNA (for example, from our aardvark library) has been isolated, it is routinely sequenced. In four separate tubes, the aardvark DNA is added to a DNA replication mixture containing nucleotides, DNA polymerase, and... A labeled dideoxynucleotide. Each tube gets a different dideoxynucleotide, either ddA, ddT, ddC, or ddG When a dideoxynucleotide gets added during DNA replication, it causes chain termination, which means that replication stops The labeled fragments from the four mixtures (corresponding to A, T, C, and G) are separated by size using polyacrylamide gel electrophoresis) By arranging the fragments in order of size, the sequence of the DNA is determined. In automated sequencers, a single mixture is used, with different fluorescent labels for each nucleotide, and the fragments are separated by capillary electrophoresis and analyzed automatically A DNA sample can be amplified in a test tube without the need for cloning, using the Polymerase Chain Reaction (PCR) technique The DNA is replicated using a thermostable DNA polymerase (for example, the Taq polymerase from Thermus aquaticus) with alternating rounds of heating and cooling in a device called a thermal cycler Since DNA polymerase requires a short segment of DNA to begin replication (a primer), the exact sequence amplified can be controlled by choosing the appropriate primer This technique can be used with a very small starting sample – for example, the DNA from a single hair strand at a crime scene The use of genomic sequence databases to find genes and to predict their behavior Closely connected with Genomics, the analysis of the complete genomic sequence of a species; and Proteomics, the analysis of all the proteins made by a species