Manipulating DNA

1. Manipulating DNA • Recombinant DNA: a combination of DNA in ways that “nature never intended” • Usually, DNA from different organisms combined • Terminology • Clones: organisms, cells, or molecules that are identical copies (in this case, usually DNA) • Tools: restriction enzymes, vectors • Procedures: gel electrophoresis, Southern blot, PCR, bacterial and cell culture methods

2. Restriction enzymes • Produced by bacteria to destroy virus DNA • “restriction endonucleases” • Named after bacterium, e.g. EcoR1 from E. coli • Recognize specific sequences in DNA • Recognition regardless of source of DNA • Sequences usually 6 bases, feature palindromes • Enzymes usually make staggered cuts • Produce “sticky ends” which are free to base pair with other DNA cut the same way. • Different DNA molecules cut with same enzyme can be spliced together.

3. Restriction enzymes http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif

4. Cloning • Having only a few DNA molecules is not too useful • Can’t find them on a gel • Absorb to things and get lost • Destroyed by stray enzymes • Carrying out genetic manipulation requires multiple copies of the same molecule • Identical copies: clones • Naked pieces of DNA are easily lost or destroyed by enzymes and cannot be replicated. • Insertion into a “vector” solves those problems.

5. Cloning and vectors • Traditional cloning: to get multiple copies of DNA of interest, must get it into a cell • Vector: means for moving DNA into a cell where multiple copies can be made • Vectors may be plasmids • Easily shuttled into bacteria or plants (Ti plasmid) • Plasmids are replicated within cells • Vector may be a bacteriophage • Protein coat in which DNA is packaged, then delivered

6. Plasmid vectors • Plasmids are circular DNA molecules • Plasmids can be cut with restriction enzymes and piece of DNA inserted • Plasmids can be taken up by bacterial cells • When cell multiplies, so does plasmid with DNA • Plasmids can multiply to high numbers in cell • Thus making multiple copies of your DNA • Bacterial clone (colony) that has your DNA must be selected for and screened for.

7. Getting a plasmid into bacteria is one thing How do you know which of the few bacteria actually took up the plasmid? How can you know your gene of interest is actually in the plasmid? (cut plasmid can re-circularize w/o insert) http://wilfred.berkeley.edu/~gordon/BLOG-images/bacteria1.jpg

8. Cloning with pUC18 Selection: bacteria that don’t have the plasmid are unwanted (and numerous). Ampicillin in culture medium kills those. Screening: Not all plasmids contain inserted DNA. Beta-galactosidase (lac Z) turns “X-gal” blue; insert in lac Z gene = white colonies. http://www.isu.edu/~shiemalc/courses/Molecular%20Biotechnology/Images/pUC18.gif

9. Screening with X-gal www.mun.ca/.../scarr/ Blue_&_White_Colonies.html

10. Using phage • DNA can be cloned into Bacteriophage • When virus replicates, makes multiple copies of its DNA including the DNA to be cloned • Plaque contains new viruses and cloned DNA http://www.tk2.nmt.ne.jp/~czar/phage_gif/plaq_p7.jpg

11. YAC • Yeast artificial chromosome • Has a centromere, telomeres, restriction sites, and lots of room to put cloned DNA in. • Carries very large pieces, good for eukaryotic DNA libraries • A set of clones containing most to all of an organism’s genome • DNA cut with restriction enzymes, cloned; individual clones have to be identified later. • Yeast = eukaryote and safe; good for making proteins, especially glycoproteins.

12. YAC structure YAC starts circular. Contains a centromere, an origin to allow its replication, and two BamH1 sites that open up the circle leaving telomeres at the end. DNA can be cloned into a restriction site (e.g. EcoR1). http://www.inrp.fr/Acces/biotic/biomol/outilsbm/images/yac4.gif

13. PCR: identification, amplification, or cloning of DNA through DNA synthesis DNA synthesis, whether PCR or DNA replication in a cell, is carried out by DNA polymerase. DNA polymerases have three requirements: Only work in a 5’ to 3’ direction Require a template: DNA to copy. Require a primer: a free 3’ OH end to add to. PCR uses Taq polymerase, a heat stable polymerase from the thermophilic bacterium Thermus aquaticus.

14. Facts about PCR • PCR requires sequence information • Primers on either side of area of interest must be complementary, thus you must know sequence. • Works best on fairly small fragments of DNA • Theoretically, can work on a single DNA molecule. • This is where PCR really shines. • Is a method for amplification: make enough DNA to see a band on a gel; substitute for traditional cloning.

15. PCR Template DNA Primers dNTPs Taq polymerase • Denature (high heat) • Anneal primers • Synthesize • Repeat http://www.pitt.edu/~heh1/PCR.jpg

16. Some PCR details • Primers: 18-22 nucleotides long, typically • Geometric increase in DNA: from 1 molecule, 34 million in 25 cycles. • 5000 bp limit in size • Most reliable when starting with at least 10-100 DNA molecules. • http://faculty.plattsburgh.edu/donald.slish/PCRmov.html • MANY animations online.

17. Making a genomic library • DNA from organism is cut with restriction enzymes; vector DNA (e.g. YAC) also cut. • Restriction fragments inserted into vectors • Vectors inserted into cells which grow • Multiple copies of DNA obtained • Library of expressed DNA using cDNA • If expressed, then can be collected as mRNA • Reverse transcriptase can make DNA from RNA template

18. Action of reverse transcriptase Poly-T synthetic oligonucleotide acts as primer, mRNA is the template. DNA-RNA hybrid is denatured, RNA is digested away, and DNA polymerase makes a ds DNA of the ss DNA. This piece of DNA, representing an active gene, can now be cloned.

19. Finding a clone • Finding a clone anywhere involves using a probe. • A probe is a nucleic acid that is sufficiently complementary to the DNA of interest that it will base-pair with it. • The probe must be labeled or else the binding will be invisible (and thus worthless) • Labeling is usually with radioactive isotopes or fluorescent compounds • Detection is with x-ray film

20. DNA hybridization • Use of a probe results in a ds molecule where the strands are from different sources: hybrid. • Heat (or alkali) required to denature DNA (ss) • Done at high salt • Na+ stops PO4- from repelling each other • Done at elevated temperature • Incorrect (weak) matches between strands come apart, allow correct ones to form. • Complementary strands “zip up” once they find each other. • If you have primers, you can use PCR instead to find your DNA: the one that gets amplified.

21. Colony (in situ) hybridization • For finding e.g. bacterial colonies that contain your clone. • Cells in colonies on a plate are lysed, the DNA is denatured and transferred to a membrane. • Membrane is mixed with probe (complementary and labeled) • Loose probe is washed away • Membrane covered with x-ray film • Colony with clone of interest makes spot on film

22. Characterizing cloned DNA • Restriction maps • In this example, a 1650 bp clone is cut by SalI into 2 pieces, large and small. • Cutting with KpnI makes 3 pieces, so 2 sites must be present. • Combination of enzymes allow pieces to be put in order. http://www.umanitoba.ca/faculties/afs/plant_science/courses/39_314/l07/map2digest.gif

23. Southern Blot

24. DNA sequencing • DNA sequencing is a method that reveals the sequence of nucleotides in a stretch of DNA. • Sequencing requires DNA synthesis; you actually determine the sequence of a complementary strand. • A template and primer are mixed with a polymerase, dNTPs, and a smaller proportion of ddNTPs

25. DNA sequencing-2 ddNTPs (Dideoxy NTPs) are missing an -OH group at the 3’ position. Remember that this is where the next nucleotide must attach during DNA synthesis. • There are many DNA molecules in a test tube, all being copied at the same time. If a ddNTP is inserted into the growing DNA strand, synthesis of that molecule STOPS. http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/dideoxy.jpg

26. DNA sequencing-3 • ddNTPs are inserted at random (that is, when G is called for, sometimes the enzyme will grab ddGTP) • This process results in a collection of DNA molecules of different lengths that can be separated on a gel. • Since all 4 ddNTPs are present, growth of the DNA can stop after any base, producing a large number of DNA molecules that differ in size by 1 base. • Each ddNTP is fluorescently labeled with a different color. The whole mixture is separated in a run-off gel, and the different colors marking which base terminated the reaction can be detected. http://www.dnalc.org/ddnalc/resources/shockwave/cycseq.html

27. DNA sequencing-4 This picture shows a few of the different sized molecules that can result. Notice that each ends with a particular ddNTP; in modern practice, this will have a color associated with it and thus be identified. derived from opbs.okstate.edu/.../ img015.jpg

28. DNA sequencing gel Inexpensive, older method: Four reaction tubes, each containing a different ddNTP: ddATP, ddCTP, etc. Contents of each tube run in a different lane, stained to see the bands.Gel read from bottom up. carnegieinstitution.org/.../ sequencinggel.html

29. DNA sequencing w/ fluorescent labels ddCTP = blue ddATP = green ddTTP = red ddGTP = grey medlib.med.utah.edu/.../biochem/ Formosa/menu.html