1. Updated Summer 2015 Jerald D. Hendrix

2. A. Recombinant DNA Technology 1. Restriction Endonucleases 2. Creating a Recombinant DNA Library 3. Properties of a Cloning Vector 4. Screening a Recombinant DNA Library 5. DNA Sequencing 6. Polymerase Chain Reaction 7. Bioinformatics

3.  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!)

4.  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

5.  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.

6.  Type II Restriction Endonuclease (cont.)  Example: Eco RI 5’ ↓ 3’ -N-N-G-A-A-T-T-C-N-N- -N-N-C-T-T-A-A-G-N-N- 3’ ↑ 5’ 5’ 3’ -N-N-G A-A-T-T-C-N-N- -N-N-C-T-T-A-A G-N-N- 3’ 5’

7.  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

8.  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)

9.  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”

10.  Creating a Recombinant Library (continued)  Plate the transformed E. coli onto X-gal (5-bromo-4- chloro-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!

11.  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. amp R ). By using medium containing the antibiotic (ampicillin), only cells transformed with the vector DNA can survive. This selects against untransformed cells.

12.  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 β - galactosidase.  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.

13.  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

14.  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

15.  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

16.  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.

17.  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

18.  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

19.  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