2.2 analyzing and manipulating dna

PowerPoint for BI 520-01
Spring 2013

1. Analyzing & Manipulating DNA

2. What is genetic engineering? Genetic engineering, also known as recombinant DNA technology , means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism ; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.

3. Special Cases: Selective Breeding Artificial selection : breeding only those plants or animals with desirable traits People have been using selective breeding for 1000’s of years with farm crops and domesticated animals.

4. Selective breeding

5. Hybridization The crossing of dissimilar individuals to bring together the best of both organisms, but genomic incompatibility usually leads to sterility.

6. Or does it?  While there is no known instance of a male mule siring offspring, female mules have on very rare occasion given birth to viable offspring .   Most of the offspring of these female mules have been sterile, except on stallion that was completely reverted. male horse  + female donkey Donkey Horse Mule Hinny   female horse   +   male donkey   female mule   +   male donkey = sterile offspring   female mule   +   male horse = male stud

7. Genetic Bottlenecks & Founder Effects Can be likened to Artificial Selection, hybridization, and inbreeding

8. Inbreeding The continued breeding of individuals with close relation to one another. Inbreeding rapidly purifies the genome by exposing homozygous recessives.

9. Genetic Engineering

10. Basic steps in genetic engineering Isolate the gene Insert it in a host using a vector Produce as many copies of the host as possible Separate and purify the product of the gene Manipulate it any way you want

11. Vectors are our canvases Basic vector musts: 1 Origin of Replication ( phyllum specific) 2 Ability to select for/against 3 Multiple cloning site

12. Cloned Enzymes!!! Polymerase – make new DNA or RNA against an existing DNA or RNA template Ligase – joins two DNA or RNA Nuclease – cleaves into two DNA Repair Proteins – self-explanatory Methyltransferase – adds methyl groups Phosphatase - removes 5´ phosphate groups from DNA and RNA Kinase – adds 5´ phosphate Recombinase – Swap strands in and out

13. Polymerases copy DNA and RNA Taq  DNA Polymerase is thermostable polymerase that elongates chains 5´→ 3 ´ Klenow Fragment goes 5 ´→ 3´ T7 RNA Polymerase synthesizes RNA in the 5´→ 3´ direction from DNA template containing a T7 phage promoter.

14. Ligases glue pieces together

15. Exonuclease I – binds single-stranded; removes primers in a polymerase reaction mixture Exonuclease III – binds double-stranded; chews back 3´-5´; can control the number removed. Nucleases cut DNA

16. Type I endonucleases were the discovered first. bind at one place but cut a random distance (c.a.1000 bp ) away. Cleavage follows a process of DNA translocation, which shows that these enzymes are also molecular motors. asymmetrical recognition site is and is composed of two specific portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a non-specific spacer of about 6–8 nucleotides. These enzymes are multifunctional and are capable of both restriction and modification activities, depending upon the methylation status of the target DNA . Nuclease Endonucleases

17. TYPE II endonucleases Aside from PCR and primers, ‘restriction enzymes’ are the major workhorses in our toolkit.

18. Most RE Recognition Sequences are Palindromes G^AATT-C C-TTAA^G G^GATC-C C-CTAG^G A^GATC-C T-CTAG^G GC^GGCC-GC CG-CCGG^CG Eco RI Bam HI Bg1 II Nof I

19. More RE Enzymes Enzyme Sequence Product Eco RI G^AATTC 5’ sticky ends Bam HI G^GATCC 5’ sticky ends Bg1 II A^GATCT 5’ sticky ends Pvu I CGATC^G 3’ sticky ends Pvu II CAG^CTG Blunt end Mbo I G^ATC 5’ sticky ends Hind III A^AGCTT 5’ sticky ends Hinf I G^ANTC 5’ sticky ends Sau 3A G^ATC 5’ sticky ends Alu I AG^CT Blunt end Taq I T^CGA 5’ sticky ends Hae III G^GCC 5’ sticky ends Nof I GC^GGCCGC 5’ sticky ends

20. DNA Fingerprinting

21. Gel Electrophoresis (1/2)

22. Gel Electrophoresis

23. Gel Electrophoresis (2/2)

24. Methyltransferases Phosphatases Kinases Recombinases

25. PCR – Polymerase Chain Reaction Making copies to work with.

26. PCR Cloning Primer Design Specific primers Degenerated primers Nested primers Amplification High-fidelity DNA polymerase Hot start Touch down PCR Clone into appropriate vector Compatible restriction sites Poly T (pGEM T easy) No ligation (Topo cloning)

27. Primer Design Considerations Primers must be specific for desired sequence to be amplified primers should be long enough to ensure specificity (usually 18-30 bases) primers normally screened against databases Primers must form stable duplex at annealing temperature No complementarity between forward and reverse primers or primers and product

28. Initial primer selection criteria Length (18-25 bases) Base composition (45-55% GC) Melting temperature (55-80  C) 3’ terminal sequence strong bonding base (G or C) at end no runs (3 or more) of G or C at end

29. Primer complementarity criteria Primer vs. self & forward vs. reverse maximum number of consecutive bonds maximum number of consecutive G-C bonds Forward primer vs. Reverse primer maximum number of consecutive bonds between the 3’ ends Primer vs. product maximum number of consecutive bonds between the 3’ ends

30. PCR amplification PCR amplification cycles consist of 3 main steps: a. Denaturing step leading to strand separation which occurs at high temperature (usually over 80°C and typically 94-95 °C ). The denaturing step usually lasts for 15 to 30 sec but can be extended for long fragments of template DNA and Hot Start Polymerases ( 2-3 min ). b. Annealing step corresponds to primer hybridization with the template. The annealing temperature is dependent on the primer sequences and base composition and usually is kept lower than 72°C. Annealing time of 30-45 seconds commonly used. Increasing annealing time does not drastically influence the outcome of PCR reactions- DNApol has reduced activity @ 45-65°C, thus longer annealing times may increase the likelihood of nonspecific amplification. Tm of primer in °C = (no. of Gs and Cs x 4) + (no. of As and Ts x 2) Ideally the Tm of each primer (forward and reverse) should be similar (±2 °C) The optimal annealing temperature to reduce NSB is usually (Tm – 5 °C) c. Extension step is normally 72°C for NPRs and 68 °C for PRs; duration normally 1 min for every 1 Kb to be amplified (depends on the DNApol used ). *Usually a final extension at 72°C for 5 to 10 min is included at the end of the cycle to allow the completion of the extension of all the products.

31. What if all you know is the protein sequence?

32. Degenerate Primers

33. Example a protein motif:       W D T A G Q E Trp Asp Thr Ala Gly Gln Glu 5'  TGG GA Y AC N GC N GG N CA R GA R   3'    where the Y = C or T, R = G or A, N = G, A, T or C. (This gives a mix of 256 different oligonucleotides.)

34. What if you don’t even know the protein sequence?

35. Degenerate Primers for Discovery P P P P P P P

36. Degenerate Primers

37. Degenerate Primers for Discovery P P P P P P P V /A V/S K P L V/G P A S GUN GUN AAA CCN UUA GUN CCN GCN AGU GCN UCN AAG UUG GGN AGC CUN GYN KYN AAR CCN YTN GKN CCN GCN AGY 24 224 2 4 2 4 24 4 4 2 = 262,144 SEQUENCES!!

38. Some genes this is already done

39. Touch Down / Step down A one-step procedure for optimizing PCRs 45- 55 55- 45 55 It involves the use of an annealing temperature that is higher than the target optimum in early PCR cycles. The annealing temperature is decreased by 1°C every cycle or every second cycle until a specified or 'touchdown' annealing temperature is reached. This only works on paralogs if there are sequence differences in the “primed” sequence.

40. NESTED PCR A powerful method to amplify specific sequences of DNA from a large COMPLEX mixture of DNA. Overcomes non-specific amplification [even paralogs * ] by using two sets of primers (almost like using a longer primer). * Primer Design is crucial!

41. DNA Sequencing By separation (Sanger) By synthesis Polymerase vs. Ligase fluorophore vs. pyrophosphate Polony vs. multiplexing Nanopore

42. PCR-based Dye terminator nucleoside Massive capillary electrophoresis Sanger Sequencing

43. 454 FLX

44. Pyrosequencing

45. IlluminaSolexa Sequencing

46. Nanosequencing

47. SMRT Sequencing