Cloning and Sequencing

1. Cloning and Sequencing

2. Project overview

3. Background • Project will have you cloning the gene that codes for the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) • GAPDH is a housekeeping gene necessary for survival • GAPDH is an enzyme that is crucial for glycolysis to occur

4. Glycolysis

5. GAPDH • can be easily isolated in cells • Is made up of four subunits that are either identical (homotetramer) or in pairs of slightly different proteins (heterodimer) • Has two domains: amino terminal region binds to NAD+ while the carboxy terminal region has the dehydrogenase activity • Does two things: • Removes H+ from GAP and transfers it to NAD+ • Adds second Phosphate to GAP

6. GAPDH genes • Found in the cytosol (glycolysis) and in the chloroplast as part of photosynthesis • Isozymes coded for on nuclear DNA • GAPC denotes the gene that codes for cytosolic GAPDH and is the gene that we will study. • The GAPC protein is a heterodimer.

7. Gene Cloning

8. Big picture for this unit • Isolate GAPC gene from plants • Amplify the GAPC gene by nested PCR • Assess the results of the PCR • Purify the PCR product containing GAPC • Ligate (insert) GAPC gene into plasmid vector • Transform bacteria with new plasmid • Isolate plasmid from bacteria • Confirm plasmid by restriction digests • Prepare plasmid DNA to be sequenced by outside facility • Analyze sequence of your GAPC gene using bioinformatics

9. Nucleic Acid Extraction • Task is to separate DNA from rest of the cellular components, including membranes, proteins, and enzymes • Must also remain in tact after extraction • Plant cells also have a cell wall to disrupt • Nucleases can digest DNA • Acidic contents of organelles can damage DNA • Some plants have polyphenols that bind to DNA rendering it useless for experiments

10. Basic Steps of DNA Extraction • Harvest cells from fresh, young plants • Grind cells to physically disrupt tissue & cell walls • Lyse cells to disrupt membranes • Remove cellular debris by centrifugation • Digest remaining cellular proteins

11. Basic Steps of DNA Extraction • Purify DNA by ion-exchange chromatography to remove contaminants • Concentrate DNA by ethanol precipitation • Determine purity and concentration of DNA with UV Spec

12. Lysis Buffers • EDTA to destabilize the membrane and inhibit nucleases • Buffers to maintain pH since acids are released by organelles • Detergent to dissolve membrane • DTT denatures proteins

13. Polymerase Chain Reaction • Rapidly creates multiple copies of a segment of DNA • Uses repeated cycles of DNA synthesis in vitro • Used in DNA fingerprinting, kinship analysis, genetic testing for mutations, and infectious disease for diagnosis

14. PCR Round 0 = 1 copy Round 35 = billions of copies

15. PCR players • DNA template – targeted piece of DNA • Primers – small segments of DNA that bind complementary upstream and downstream of the target on the template • Taq DNA polymerase – isolated from the Thermus aquaticus bacteria found in hotsprings of Yellowstone Park • DNA nucleotides in the form of deoxynucleoside triphosphates (dNTPs) • Reaction Buffer – maintains pH for enzymes

16. General PCR Process • Denaturation – split apart the two DNA strands by heating them to 95oC for 1 min • Annealing – primers bind to target sequence by cooling reaction to 40-60oC for 1 min • Extension – Taq Polymerase extends the primers and copies each DNA template strand by heating to 72oC for 1 min

17. Primers • Required for both sides of the target sequence (forward & reverse primer) • Length of primer is generally 18-30 nucleotides • G/C content and intra-complementarity are a concern when designing primers • Actually not a single primer for each but a mixture of primers (oligoprimers) if the sequence of the target is not known • If amino acid sequence of gene product is used then degenerate primers must be used • Initial forward primer is GABTATGTTGTTGARTCTTCWGG B=G/T/C R=G/A (purines) W =A/T

18. Nested PCR • Initial PCR primers are degenerate and based on a consensus sequence • The chances that the initial primers will bind to sequences other than the target are high • A second set of primers designed to be more specific to GAPC is used • They are nested within the initial primers and are not degenerate thus much more specific to the GAPC gene

19. Nested PCR

20. Our experiment Set-up Tube 1: negative control (no DNA) Tube 2: Arabidopsis gDNA Tube 3: Positive control pGAP plasmid Tube 4: Your plant DNA PCR Plan 1st round2nd round (nested) Initial Denaturation 95oC for 5 minutes 95oC for 5 minutes Then 40 Cycles of: Denaturation 95oC for 1 minute 95oC for 1 minute Annealing 52oC for 1 minute 46oC for 1 minute Extention 72oC for 2 minutes 72oC for 2 minutes Final Extension 72oC for 6 minutes 72oC for 6 minutes Hold 15oC forever 15oC forever

21. Gel Electrophoresis

22. PCR purification • Small impurities can have a negative effect on the ligation of the PCR product to vector DNA • Impurities include unincorporated dNTPs, polymerases, primers and small primer-dimers. • A PCR Kleen spin column will remove the impurities in less than 4 min.

23. Gene Cloning • Cloning is the production of exact copies of a piece of DNA. • It requires ligating (splicing) the PCR product into a cloning vector – often a plasmid DNA • The recombinant DNA of the ligation product can now be put into a cell to propagate (replicated)

24. Plasmids are good vectors: • small (2,000 – 10,000 bp) • circular, self-replicating • high copy number • multiple cloning sites (MCS) • selectable markers (Amp-resistance) • screening (reporter genes, positive select) • control mechanisms (lac operon) • can handle the size of the insert

25. pJet1.3 blunted vector • Designed for blunt-end cloning • High copy number • Contains Amp-resistant gene • Contains eco47IR gene which allows for positive selection • It is 2,974 bp long

26. Inserts • Sticky ends have single strands of nucleotides on ends and are good for directional inserting • Blunt ends have no single strands and thus are easier to insert but are non directional.

27. Ligation • T4 DNA Ligase catalyzes formation of phosphodiesterase bond between 3’ hydroxy on one piece and the 5’ phosphate on another piece. • Requires ATP and Mg+2 • Insert to vector DNA ratio should be 1:1 • Proofing reading DNA polymerase removes dangling 3’A of PCR product

28. Products of Ligation • Self-ligation of vector • Ligation of vector to primer-dimers • Ligation of multiple inserts • Self-ligation of inserts • Ligation of one insert into vector

29. Transformation • Once PCR product (insert) has been ligated into a plasmid, the plasmid be introduced into a living bacterial cell to replicate. • Two methods of transformation: • Electroporation • Heat Shock • Both methods make cells competent - able to take up plasmids

30. Transformation Steps • Wash away growth media from cells • Place cells in ice cold calcium chloride which most likely hardens the cell membrane • Add plasmid to cells • Move cells to hot environment (usually 42oC) causes membrane pores to open so plasmid can enter • Add nutrient media to cells to allow them to recover from stress • Plate cells on selective growth plates (Amp and IPTG (increases expression of ampr gene)

31. Microbial Culturing • Pick a colony from the transformed cells to innoculate a liquid culture • Liquid culture (broth) must have selective antibiotic (Amp) in it. • Choose a single colony from the plate • Under favorable conditions, a single bacteria divides every 20 minutes and will multiply into billions in 24 hours

32. Plasmid Purification • To confirm that the engineered cells have been transformed with the correct DNA • Different methods • Lysozyme Method • Alkaline Cell Lysis Method • Column Methods (Aurum)

33. Plasmid preps • Spectrophotometer determination of culture density. Take OD600 of culture (equal to about 8x108 cells/ml • Aurum column can process up to 12 OD●ml of bacterial host cells • Cells disrupted with a lysis buffer • DNA binds to membrane of column, is washed and then eluted with aqueous buffer.

34. Restriction Digests • DNA cut with restriction enzymes • Evolved by bacteria to protect against viral DNA infection • Endonucleases -cleave within DNA strands • Over 3000 known enzymes

35. Restriction Digests • Each enzyme cuts DNA at a specific sequence= restriction site • Many of the restriction sites are 4 or 6-base palindrome sequences Enzyme cuts Fragment 2 Fragment 1

36. Enzyme Examples EcoRI G-A-A-T-T-C C-T-T-A-A-G HindIII A-A-G-C-T-T T-T-C-G-A-A BamHI G-G-A-T-C-C C-C-T-A-G-G Bgl II A-G-A-T-C-T T-C-T-A-G-A

37. Restriction Digest • Restriction Buffer provides optimal conditions: • NaCl provides correct ionic strength • Tris-HCl provides proper pH • Mg+2 is an enzyme co-factor • Body temperature (37oC) is optimal • Too hot kills enzyme • Too cool takes longer digestion time

38. DNA Sequencing • Determining the exact order of the nucleotide sequence in a DNA molecule. • Use to take days, now takes hours • Have sequences of entire genones for over 700 organisms

39. Sanger Method • Prepare single-stranded DNA template to be sequenced • Divide DNA into four test tubes • Add primer to each tube to start DNA synthesis • Add DNA polymerase • Add labeled deoxynucleotides (dNTP) in excess. Labeled with radioactive or fluorescent tags • Add a single type of dideoxynucleotides (ddNTPs) to each tube. When incorporated in sythesized strand, synthesis terminates. • Allow DNA synthesis to proceed in each tube • Run newly synthesized DNA on a polyacrylamide gel

40. Reading the Sequence • In the tube with the ddTTP, every time it is time to add a T to the new strand, some Ts will be dTTP and some will be ddTTP. • When the ddTTP is added, then extension stops and you have a DNA fragment of a particular length. • The T tube will, therefore, have a series of DNA fragments that each terminate with a ddTTP. • Thus the T tube will show you everywhere there is a T on the gel • Same thing happens in all tubes • Read gel from top to bottom looking at all four lanes to get the sequence.

41. Automated Sequencing • Dye-terminator sequencing labels each of the ddNTPs with a different color fluorescent dye. • Now reaction can be run in one tube • Use capillary electrophoresis rather than the standard polyacrylamide slab gel. • When DNA fragment exits gel, the dyes are excited by a laser and emit a light that can be detected . • Produces a graph called a chromatogram or electopherogram

42. Automated Sequencing

43. Bioinformatics • Computerized databases to store, organize, and index the data and for specialized tools to view and analyze biological data • Uses include • Evolutionary biology • Protein modeling • Genome mapping • Databases are accessible to the public • Allow us to record, compare, or identify a DNA sequence