Techniques in Cloning

1. Techniques in Cloning

2. 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

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

4. 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

5. General PCR Process • Denaturation – split apart the two DNA strands by heating them to 95oC for 30s -1 min • Annealing – primers bind to target sequence by cooling reaction to 40-60oC for 30s - 1 min • Extension – Taq Polymerase extends the primers and copies each DNA template strand by heating to 72oC for 30s - 1 min

6. 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

7. 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 target is used • They are nested within the initial primers and are not degenerate thus much more specific to the target gene

8. Nested PCR

9. Our experiment Tube setup: Add the following to a P3 tube (with PCR reaction pellet) 5 ul Target DNA template (P1) 10 ul Primer set (P2) 15 ul Enzyme -grade water (P4) PCR Plan Initial Denaturation 94oC for 5 minutes Then 30 Cycles of: Denaturation 94oC for 30 sec Annealing 50oC for 30 sec Extention 72oC for 30 sec Final Extension 72oC for 5 minutes Hold 15oC forever

10. Gel Electrophoresis • Definition: the process of separating molecules based on size and charge • Agarose: highly purified agar, heated and dissolved in buffer. Forms a matrix of pores for molecules to travel through. • Smaller molecules travel further • Molecules migrate towards the positive (red) end of the chamber

11. Gel Electrophoresis • Process • Make Agarose gel • Thinner gels (0.8%) yield better results for larger DNA • Prepare samples • Restriction enzymes used to cleave at specified sites • Apply samples to gels, apply current • If samples run from positive end they will run off the gel • Stain gels to see bands • Would not be able to see bands if we did not stain

12. Gel Electrophoresis • DNA molecules have a negative charge • This allows them to migrate towards the positive end of the chamber • The samples and the electrophoresis chamber use specialized buffers. Using TAE/TBE buffer helps stabilize the sample and allows the reaction to occur quicker in the chamber. • If water were in the chamber instead of TAE/TBE buffer the reaction would take much longer or migration may not occur at all • Stains: ethidium bromide will cause the bands to glow orange under UV light. Fast stain will result in blue bands

13. Uses for Gel Electrophoresis • DNA fingerprinting or profiling • Paternity testing • Crime scene sample analysis • Identification of bacteria and other pathogens • Who is credited with discovering the DNA profiling process? • Alec Jefferies in 1985

14. Gel Electrophoresis

15. 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 spin column will remove the impurities in less than 4 min.

16. Restriction enzymes (endonucleases) • Definition: class of enzymes that cleave (cut) DNA at a specific and unique internal location along its length. • Makes 2 incisions, one through each of the sugar-phoshate backbones of the double helix • They can be naturally produced in bacteria and the bacteria use them as a defense mechanism against viral infection • The enzymes chop up the viral nucleic acids and destroy the virus • More than 3,000 known restriction enzymes • Common ones are: EcoRI, Psti, HindII

17. Restriction enzymes (endonucleases) • Discovered in late 1970s by Arber, Smith and Nathans • The chemical bonds that the enzymes cleave can be reformed by other enzymes known as ligases • Uses: • To insert new segment of DNA • To cut specific segments of DNA to study • To cut segment from one gene to insert it into another • Genetic engineering or recombinant DNA • Need suitable host, vector for carrying plasmid, way to get host to take up gene

18. 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

19. 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

20. Restriction enzymes (endonucleases) • Sticky ends: when unpaired length of bases (5’ AATT 3’) encounter an unpaired length of sequences (3’ TTAA 5’), they will bind or are “sticky” for each other. • Blunt ends: same length sequences or DNA section cut in half • Joining of two blunt ends is ligation

21. 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 • Specific enzymes have specifc temps and times

22. 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

23. 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

24. Bacterial DNA Bacterial cell Plasmid DNA Genomic DNA

25. 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

26. 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

27. 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)

28. 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

29. 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, EZNA)

30. Plasmid preps • Spectrophotometer determination of culture density. Take OD600 of culture (equal to about 8x108 cells/ml • 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.

31. 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

32. Lambda DNA • Lambda DNA comes from a bacteriophage • Genomic DNA of Lambda is well studied and used in research as a size markers for DNA pieces • Arrow mark HindIII restriction sites

33. 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

34. 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

35. 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.

36. 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

37. Automated Sequencing

38. 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

39. Types of RNA • Messenger RNA (mRNA) • Tranfer RNA (tRNA) • Ribosomal RNA (rRNA) • Signal Recognition Particle RNA (SRP RNA) • Small Interfering RNA (siRNA) – gene reg • Micro RNA (miRNA) – gene reg.

40. RNA Interference (RNAi) • Dicer enzyme cuts dsRNA up into smaller siRNA which then complex into the RNA-induced silencing complex (RISC) which then cuts up the mRNA • dsRNA can be engineered so that genes can be systematically shut down

41. Reverse Transcriptase PCR • Use reverse transcriptase to make a DNA copy of mRNA • Copy called cDNA • Allow scientists to study the level of gene expression in a cell

42. Northern Blots • Run mRNA on a gel • Transfer it to nitrocellulose membrane • Add labeled cDNA probes to the membrane and hybridize the probes to the RNA • Allows you see what genes are expressed

43. DNA Microarray (Chip) • Adhere genes to chip • Collect mRNA from cells • Make labelled cDNA from mRNA (red + green) • Add cDNA to chip • Measure signal