Mastering Restriction Endonucleases in Recombinant DNA Library Creation

1. Molecular DNA Biotechnology Updated Summer 2015Jerald D. Hendrix

2. Microbial Biotechnology • Recombinant DNA Technology • Restriction Endonucleases • Creating a Recombinant DNA Library • Properties of a Cloning Vector • Screening a Recombinant DNA Library • DNA Sequencing • Polymerase Chain Reaction • Bioinformatics

3. Restriction Endonucleases • 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. Restriction Endonucleases • 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. Restriction Endonucleases • 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. Restriction Endonucleases • 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-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. Recombinant DNA Library • 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. Recombinant DNA Library • 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. Recombinant DNA Library • 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. coliin 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. Recombinant DNA Library • 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. Properties of a Cloning Vector • 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.

12. Properties of a Cloning Vector • 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. Screening a Recombinant Library • 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. Screening a Recombinant Library • 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. Screening a Recombinant Library • 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. Screening a Recombinant Library • 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. DNA Sequencing • 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. Polymerase Chain Reaction • 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 Thermusaquaticus) 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. Bioinformatics • 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