DNA Technology

1. DNA Technology

2. DNA Technology • Genetic Engineering - manipulating the genes of organisms Basic - centuries old – selective breeding NEW - recombinant DNA technology - mixing of genes from two or more sources and incorporating them together

3. Common Practice: insert a gene from one source into bacteria and culture the bacteria so it reproduces the gene over and over - gene is removed and placed into a plasmid (small circular DNA) and then the plasmid is put back into the bacteria (transformation) - bacteria reproduces- copies the plasmid and codes for the gene put in the plasmid POINT: cloning a gene for use of the protein product or use the gene itself

4. Bacterium Cell containing geneof interest Plasmid Gene of interest Bacterialchromosome DNA ofchromosome RecombinantDNA (plasmid) Recombinatebacterium 3 Gene of interest Protein expressedby gene of interest Copies of gene Protein harvested Basic research on protein Basic research on gene Gene used to alterbacteria for cleaningup toxic waste Human growth hormone treatsstunted growth Gene for pestresistance inserted into plants Protein dissolvesblood clots in heartattack therapy

5. HOW? Restriction Enzymes - enzymes that cut DNA in a specific place and a specific way Place: Restriction Site - usually symmetrical segment of DNA ---CGGTACCG--- ---GCCATGGC--- Cut: Staggard - leaves two short single strands of DNA = STICKY END ---C GGTACCG--- ---GCCATGG C--- DNA Restriction Animation

6. Restriction site 5 3 DNA G A A T T C 3 5 C T T A A G A A T T C G C T T A A G Sticky end A A T T C G G C T T A A Fragment from differentDNA molecule cut by thesame restriction enzyme G A A T T C A A T T C G C T T A A G G T T A A C One possible combination Recombinant DNA molecule

7. Source of Restriction Enzymes: bacteria – restriction enzymes act as an immune system and cut up DNA of invading viruses protects own DNA by methylation (changes shape of DNA so it doesn’t fit in the active site of the enzyme) Each Restriction Enzyme cuts DNA at a specific sequence - restriction sequences repeat many times in a genome so the enzyme is going to cut the DNA into many pieces = Restriction Fragments

8. Recombination Process • Find a gene of interest • Isolate DNA and expose to restriction enzyme • Isolate Plasmid and expose to SAME restriction enzyme - cuts the plasmid in the same way - same sticky ends • Mix DNA fragments and plasmids together - sticky ends match and bond using hydrogen bond = formed a CLONING VECTOR (DNA that can carry foreign DNA into a cell and replicate)

9. Seal DNA backbones with DNA ligase • Put cloning vector into solution of bacteria and allow them to take in the bacteria by transformation. • Grow bacteria • Test colonies for desired gene or gene product (PAGE 369) • Isolate colony and grow.

10. USING EUKARYOTIC DNA IN PROKARYOTES 1. Different Promoters: scientists include an EXPRESSION VECTOR - prokaryotic promoter just above eukaryotic gene - allows the Prokaryotic polymerase to bind and then move across eukaryotic gene

11. USING EUKARYOTIC DNA IN PROKARYOTES 2. Introns and mRNA modification: prokaryotes lack modification mechanisms -Solution: Make Complementary DNA (cDNA) - allow eukaryote to make mRNA and isolate it - use reverse transcriptase to make mRNA into DNA - DNA now free of introns - isolate and place in a plasmid

12. USING EUKARYOTIC DNA IN PROKARYOTES 3. Use eukaryotic cells instead of prokaryotic - make artificial chromosomes and include necessary parts - replication site - telomeres and centromeres - foreign DNA - Eukaryotic Electroporation: shock eukaryotic cells to stimulate DNA uptake - DNA guns and DNA needles – inject DNA into the cell and hope for cross over

13. Polymerase Chain Reaction CLONING DNA - Artificially (without a cell) = Polymerase Chain Reaction - page 391 - Requirements: DNA section of choice (cloning, analysis, forensics) four nucleotides heat resistant DNA polymerase DNA primers (artificially produced - function as RNA primers)

14. PCR Process: • DNA put in reaction chamber • Heated to separate strands • Cooled - primers bind • Polymerase builds new DNA • Primers stay attached • Repeat PCR Animation

15. PCR Applications: amplify DNA for recombination analyze ancient DNA forensics genetic testing

16. Gel Electrophoresis separating the DNA in a gel using an electrical charge Requirements: Separating media (gel) Electric Current Restriction Enzyme Cleaved DNA

17. Gel Electrophoresis Process: • Expose DNA to a restriction enzyme • Cuts DNA into restriction fragments • Place DNA in the Gel • Run the Current through the Gel • DNA separates according to the size of the DNA fragment • Results in a pattern of lines on the gel - each line being a different size of fragment of DNA

18. Gel Electrophoresis How: DNA has a negative charge on the phosphate groups - moves toward the positive end of the current Gel is made of polymer fibers that inhibit movement of the DNA bands - the larger the fragment the less it will move DNA is either stained or marked with a radioactive tag to make it visible Electrophoresis Animation

19. Forensics • DNA sample • Extract DNA • PCR Sample • Restriction Enzyme and radiometric/florescent nucleic acid tags – bind to specific segments of the DNA (more specific) • Gel electrophoresis  Results

20. DNA Analysis Why It Works: - Restriction Fragment Length Polymorphisms (RFLPs) – in the non-coding portion of the DNA as well as in the coding portion, the DNA has many (poly) slight variations or forms (morphs) • These are acted on by different restriction enzymesand result in fragments of different lengths • Using multiple restriction enzymes on samples of DNA allows for a more specific analysis

21. Gene Mapping Basic Procedure: • Obtain several samples of the same DNA • Cut each sample with a different restriction enzyme • Separate the samples and isolate them • Analyze the samples and determine the sequences (SANGER METHOD) • Find where the sequences of the different samples overlap and place in order

22. 1 2 3 4 Cut the DNA from many copies of an entire chromosome into overlapping frag- ments short enough for sequencing. Clone the fragments in plasmid or phage vectors Sequence each fragment ACGATACTGGT CGCCATCAGT ACGATACTGGT Order the sequences into one overall sequence with computer software. AGTCCGCTATACGA Figure 20.13 …ATCGCCATCAGTCCGCTATACGATACTGGTCAA…

23. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC ATTCGC Restriction Enzyme #2: CGCGTAGC ATT CAGT TAGTCTAG GTAC

24. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC ATTCGC Restriction Enzyme #2: CGCGTAGC ATT CAGT TAGTCTAG GTAC

25. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC Restriction Enzyme #2: CGCGTAGC CAGT TAGTCTAG GTAC ATTCGC ATT

26. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC Restriction Enzyme #2: CGCGTAGC CAGT TAGTCTAG GTAC ATTCGC ATT

27. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC Restriction Enzyme #2: CGCGTAGC CAGT TAGTCTAG GTAC ATTCGC ATT

28. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC Restriction Enzyme #2: CAGT TAGTCTAG GTAC ATTCGC ATTCGCGTAGC

29. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC Restriction Enzyme #2: CAGT TAGTCTAG GTAC ATTCGC ATTCGCGTAGC

30. Restriction Enzyme #1: CTAGCA GTAGCTAGT GTGTAC Restriction Enzyme #2: CAGT TAGTCTAG GTAC ATTCGC ATTCGCGTAGC

31. Restriction Enzyme #1: CTAGCA GTGTAC Restriction Enzyme #2: CAGT TAGTCTAG GTAC ATTCGCGTAGCTAGT ATTCGCGTAGC

32. Restriction Enzyme #1: CTAGCA GTGTAC Restriction Enzyme #2: CAGT TAGTCTAG GTAC ATTCGCGTAGCTAGT ATTCGCGTAGC

33. Restriction Enzyme #1: CTAGCA GTGTAC Restriction Enzyme #2: CAGT TAGTCTAG GTAC ATTCGCGTAGCTAGT ATTCGCGTAGC

34. Restriction Enzyme #1: CTAGCA GTGTAC Restriction Enzyme #2: CAGT GTAC ATTCGCGTAGCTAGT ATTCGCGTAGCTAGTCTAG

35. Restriction Enzyme #1: CTAGCA GTGTAC Restriction Enzyme #2: CAGT GTAC ATTCGCGTAGCTAGT ATTCGCGTAGCTAGTCTAG

36. Restriction Enzyme #1: CTAGCA GTGTAC Restriction Enzyme #2: CAGT GTAC ATTCGCGTAGCTAGT ATTCGCGTAGCTAGTCTAG

37. Restriction Enzyme #1: GTGTAC Restriction Enzyme #2: CAGT GTAC ATTCGCGTAGCTAGTCTAGCA ATTCGCGTAGCTAGTCTAG

38. Restriction Enzyme #1: GTGTAC Restriction Enzyme #2: CAGT GTAC ATTCGCGTAGCTAGTCTAGCA ATTCGCGTAGCTAGTCTAG

39. Restriction Enzyme #1: GTGTAC Restriction Enzyme #2: CAGT GTAC ATTCGCGTAGCTAGTCTAGCA ATTCGCGTAGCTAGTCTAG

40. Restriction Enzyme #1: GTGTAC Restriction Enzyme #2: GTAC ATTCGCGTAGCTAGTCTAGCA ATTCGCGTAGCTAGTCTAGCAGT

41. Restriction Enzyme #1: GTGTAC Restriction Enzyme #2: GTAC ATTCGCGTAGCTAGTCTAGCA ATTCGCGTAGCTAGTCTAGCAGT

42. Restriction Enzyme #1: GTGTAC Restriction Enzyme #2: GTAC ATTCGCGTAGCTAGTCTAGCA ATTCGCGTAGCTAGTCTAGCAGT

43. Restriction Enzyme #1: Restriction Enzyme #2: GTAC ATTCGCGTAGCTAGTCTAGCAGTGTAC ATTCGCGTAGCTAGTCTAGCAGT

44. Restriction Enzyme #1: Restriction Enzyme #2: GTAC ATTCGCGTAGCTAGTCTAGCAGTGTAC ATTCGCGTAGCTAGTCTAGCAGT

45. Restriction Enzyme #1: Restriction Enzyme #2: GTAC ATTCGCGTAGCTAGTCTAGCAGTGTAC ATTCGCGTAGCTAGTCTAGCAGT

46. Restriction Enzyme #1: Restriction Enzyme #2: ATTCGCGTAGCTAGTCTAGCAGTGTAC ATTCGCGTAGCTAGTCTAGCAGTGTAC

47. SANGER METHOD OF DNA ANALYSIS Analysis of Unknown DNA sequence - SINGLE STRAND Cap with a radioactive primer that tags one end to determine the direction of the strand Grow in four separate vials with DNA polymerase, the 4 nucleotides and a dideoxy nucleotide of one of the four nitrogenous base Adenine Vial: GTP, CTP, TTP, ATP andddATP Cytosine Vial: GTP, CTP,ddCTP, TTP, ATP Guanine Vial: GTP,ddGTP, CTP, TTP, ATP Thymine Vial: GTP, CTP, TTP, ddTTP, ATP

48. “dd” = DIDEOXYRIBOSE - missing another hydroxyl group - does not allow for bonding on the 3’ end - chain is stopped - built into the DNA randomly so makes a whole bunch of different length chains of DNA Separate the chains (gel electrophoresis) PAGE 378 Read bands in order of length to determine the order of the nucleotides Sanger Animation Cycle Sequencing

49. Other DNA Tech Cloning: - A diploid nucleus from a somatic cell is placed in a denucleated egg cell and grown to the blastocyst (150 – 200 cells) stage and then implanted in a host mother. • The host mother does not have to be the same species as the nucleus donor • Animation • Got Clones? Video

50. Other DNA Tech DNA Chip: Series of specific selectively built DNA sequences attached to a chip that is used to scan hundreds of copies of DNA for specific sequences Animation