BIOTECHNOLOGY RECOMBINANT DNA TECHNOLOGY GENETIC ENGINEERING

1. 1 BIOTECHNOLOGY RECOMBINANT DNA TECHNOLOGY GENETIC ENGINEERING

2. 2 GENETIC ENGINEERING TECHNIQUES TRANSFORMATION BY PLASMIDS CUTTING DNA AT SPECIFIC SEQUENCES; RESTRICTION ENZYMES CUT SPECIFIC SEQUENCES, CALLED "SITES" RESTRICTION / MODIFICATION NOBEL 3. MOLECULAR CLONING FOREIGN DNA INTO VECTORS GENERATES HYBRID DNA MOLECULES NOBEL DETERMINATION OF DNA SEQUENCE NOBEL SYNTHESIS OF DNA STRANDS OF PREDETERMINED SEQUENCE POLYMERASE CHAIN REACTION- AMPLIFY "ANY" SPECIFIC DNA FRAGMENT OR GENE NOBEL

3. 3 GENETIC ENGINEERING - RECOMBINANT DNA TECHNOLOGY BIOTECHNOLOGY RESTRICTION ENZYMES RESTRICTION/MODIFICATION; FOREIGN/SELF HEMOPHILUS INFLUENZAE HINCII RESTRICTION ENZYME ESCHERICHIA COLI STRAIN R1 ECORI RESTRICTION ENZYME SPECIFIC SITES, PALINDROMES MOLECULAR CLONING HYBRID MOLECULES CONSTRUCTED IN VITRO VECTOR, TARGET (OR FOREIGN) DNA PLUS DNA LIGASE, TRANSFORMATION INTO HOST CELLS POLYMERASE CHAIN REACTION (PCR) DENATURE DNA, ANNEAL PRIMERS, POLYMERIZE WITH DNA POLYMERIZATION ENZYME, DO IT AGAIN, MANY CYCLES EXPONENTIAL AMPLIFICATION DNA SEQENCE DETERMINATION – NOT COVERED/ASSIGNED

4. 4 RESTRICTION / MODIFICATION RESTRICTION - RECOGNITION OF FOREIGN DNA NON-SELF AND CLEAVAGE BY RESTRICTION ENZYME [AT SPECIFIC NUCLEOTIDE SEQUENCES OF 4 TO 6 BASE PAIRS] MODIFICATION - CHEMICAL MODIFICATION OF "OWN" DNA [THAT IS, DNA SYNTHESIZED WITHIN "OWN" CELL] SO IT CAN NOT BE CLEAVED BY RESTRICTION ENZYME MODIFICATION IS ADDITION OF METHYL GROUP TO NUCLEIC ACID BASE BY MODIFICATION ENZYME [DNA METHYLASE]

5. 5 RESTRICTION ENZYME HincII HEMOPHILUS INFLUENZAE EXTRACT PLUS: E. COLI PHAGE T7 DNA > CUT INTO 40 SPECIFIC FRAGMENTS H. INFLUENZAE DNA > NO CUTTING CONCLUDE: ENZYME IN EXTRACT WHICH: A. RECOGNIZES AND CUTS FOREIGN DNA B. CLEAVES FOREIGN DNA AT SPECIFIC SEQUENCES CALLED "SITES" RESTRICTION ENZYMES "SITES" ARE NOT SITES IN A PHYSIOLOGICAL SENSE. THEY ARE SIMPLY 4 TO 6 NUCLEOTIDES THAT HAPPEN TO BE PRESENT IN DNA WITHIN GENES OR OUTSIDE GENES. WE CALL THEM SITES FOR CONVENIENCE.

6. 6 HincII RESTRICTION ENZYME CUTS: [ENDONUCLEASES] SYMMETRY; PALINDROME FOR EXAMPLE, E. COLI PLASMID PLASMID HincII

7. 7 ADENINE N6 METHYL ADENINE

8. 8 PLASMID FROM H. INFLUENZAE HincII WILL NOT CUT ALL GTTAAC CAATTG SEQUENCES IN CHROMO- SOME ALSO METHYLATED PLASMID MODIFIED AS "SELF" IN HEMOPHILUS INFLUENZAE CELLS

9. 9 E. COLI RESTRICTION ENZYME EcoRI "STICKY" ENDS PLASMID FROM ANY OTHER CREATURE – E.G., B. ANTHRACIS GAATTC SITES METHYLATED IN CHROMOSOME OF E. COLI COMPLEMENTARY "STICKY" ENDS

10. 10 BamHI RESTRICTION ENZYME BACILLUS AMYLOLIQUEFACIENS STRAIN H BamHI MODIFICATION ENZYME N4 METHYL CYTOSINE

11. 11 CUTTING DNA AT SPECIFIC SITES EcoRI GENES INACTIVATE EcoRI COOL SLOWLY REFORMING DNA MOLECULES: DNA LIGASE

12. 12 MOLECULAR CLONING • CONSTRUCT HYBRID DNA MOLECULE IN VITRO • TARGET - GENE OR DNA FRAGMENT OF INTEREST • PLUS A VECTOR - PLASMID, PHAGE, OR VIRUS WHICH CARRIES THE GENE OF INTEREST • INTRODUCE HYBRID DNA MOLECULES INTO HOST CELLS [BACTERIA, YEAST, ANIMAL CELLS, OR HUMAN CELLS] WHICH • REPLICATE THE HYBRID MOLECULES AND • ALLOW EXPRESSION OF THE TARGET GENE • E.G., PRODUCE A HUMAN PROTEIN

13. 13 VECTOR TARGET GENE EcoRI SITE EcoRI SITE EcoRI SITE PEN-RESISTANCE ADD EcoRI ENZYME EcoRI [AND OTHER FRAGMENTS] INACTIVATE EcoRI; MIX; ANNEAL; LIGATE

14. 14 TRANSFORM PENICILLIN-SENSITIVE BACTERIA WITH MIXTURE SELECT TRANSFORMANTS e.g., PENICILLIN-RESISTANCE DUE TO VECTOR GENE IDENTIFY TRANSFORMANTS WITH A GENE OR INTEREST (TARGET) TARGET GENE EXPRESSED IN BACTERIUM TARGET PROTEIN

15. 15 CLONING VECTORS SHOULD HAVE SEVERAL PROPERTIES: THEY SHOULD BE SMALL, EASILY ISOLATED MOLECULES THEY SHOULD HAVE ONLY ONE SITE FOR THE RESTRICTION ENZYME TO BE USED THEY SHOULD HAVE THE RESTRICTION ENZYME SITE IN SOME LOCATION OTHER THAN AN ESSENTIAL GENE THEY SHOULD CARRY SOME SELECTABLE GENE OR MARKER, LIKE TETRACYCLINE-RESISTANCE THEY MUST BE REPLICONS, THAT IS, CAPABLE OF BEING REPLICATED WHEN THE HOST CELLS GROW AND DIVIDE.

16. 16 INTRONS ARE PROBLEMS FOR CLONING HUMAN [OR HIGHER ORGANISM] GENES. INTRONS ARE TOO LONG TO PERMIT USE IN THE TEST TUBE. PURIFY MESSENGER RNA; [INTRONS ARE ALREADY SPLICED OUT] COPY mRNA INTO DNA WITH REVERSE TRANSCRIPTASE: USE THIS DNA AS TARGET CUT TARGET DNA WITH RESTRICTION ENZYME PURIFY VECTOR DNA AND CUT WITH RESTRICTION ENZYME MIX THE DNA's. INACTIVATE THE RESTRICTION ENZYME, ANNEAL, LIGATE TRANSFORM BACTERIAL HOST, SELECT TRANSFORMANTS, IDENTIFY THOSE WITH HYBRID PLASMID THESE CONTAIN c-DNA CLONE, OR COMPLEMENTARY DNA CLONE

17. 17 cDNA CLONE HUMAN mRNA [AFTER PROCESSING] 5' 3' < READING FRAME > REVERSE TRANSCRIPTASE = R.T. 5' 3' RNA 3' 5' DNA RNA DEGRADED R.T. READING FRAME RESTRICTION ENZYME cDNA CUT WITH RESTRICTION ENZYME NO INTRONS PRESENT cDNA CAN BE CLONED

18. 18 POLYMERASE CHAIN REACTION = PCR AMPLIFICATION OF SPECIFIC DNA FRAGMENT EACH CYCLE HAS THREE STEPS: DENATURE DNA; ANNEAL PRIMERS (SHORT DNA STRANDS COMPLEMENTARY TO TARGET); POLYMERIZE COMPLEMENTARY STRANDS; DENATURE 90-95oC (SEPARATE STRANDS)

19. 19 ADD PRIMERS [COMPLEMENTARY TO ENDS] [HUGH EXCESS] ANNEAL (50°C) 3' 3' POLYMERIZE COMPLEMENTARY STRANDS DNA POLYMERASE dATP, dGTP, dCTP, dTTP (72oC) NEW SYNTHESIS

20. 20 FIRST CYCLE DOUBLED AMOUNT OF DNA BETWEEN PRIMERS 20 - 30 CYCLES 106 - 107 FOLD AMPLIFICATION ~3 HRS PRINCIPAL PRODUCT

21. 21 HIV PROVIRUS HUMAN CHROMOSOME A HIV DNA SPECIFIC PRIMERS B DENATURE ANNEAL PRIMERS POLYMERIZE 30 CYCLES 5' 3' 3' 5'

22. MATERIAL ON DNA SEQUENCING WILL NOT BE COVERED AND IS NOT ASSIGNED “NORMAL”, CHAIN TERMINATOR SEQUENCING NEXT GENERATION SEQUENCING SYNTHESIS OF DEFINED SEQUENCE DNA STRANDS WILL NOT BE COVERED/ASSIGNED

27. DYE

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30. 6 SEQUENCING BY SYNTHESIS - REAL-TIME PYROPHOSPHATE SEQUENCING 1. SHEAR CHROMOSOMAL DNA, DENATURE 2. LIGATE BIOTINYLATED OLIGO, CAPTURE ON STREPTAVADIN – BEADS; 28 MICROMETER BEADS, <1 DNA STRAND/BEAD 3. EMULSIFY DNA IN BEADS CONTAINING PCR REAGENTS [ENSURES NO CROSS CONTAMINATION], AMPLIFY WITH BIOTINYLATED PRIMERS; 10 MILLION COPIES OF DNA/BEAD 4. BREAK EMULSION; DENATURE DNA; DEPOSIT BEADS IN WELLS 5. WELLS: 44 MICROMETERS DIAMETER; 55 MICROMETERS DEEP; VOLUME = 75 PICOLITERS; 480 WELLS/MM SQUARE; 1.6 MILLION WELLS ON 60x 60 MM SLIDE 6. SLIDE MOUNTED ON CCD [CHARGE-COUPLED DEVICE TO DETECT AND QUANTITATE LIGHT]. 7. REAGENTS FLOW OVER AND DIFFUSE INTO WELLS 8. ADD: DNA POLYMERASE AND deoxyATP INCORPORATION PRODUCES Ppi MEASURE AMOUNT OF Ppi IN REACTION (ADD ADENOSINE-5’-PHOSPHOSULFATE (AMP-S) PLUS ENZYME SULFURYLASE AMP-S + PPi > ATP + SULFATE

31. 7 9. MEASURE AMOUNT OF ATP ADD LUCIFERASE REACTS WITH ATP > LIGHT AND AMP + PPi MEASURE AMOUNT OF LIGHT –INDICATES HOW MANY NUCLEOTIDES (OF deoxyATP) WERE INCORPORATED 10. CLEAN EACH WELL TO REMOVE RESIDUAL ATP AND UN-INCORPORATED deoxyATP ADD ENZYME APYRASE ELIMINATES ATP; CATALYZES ATP > AMP + PPi ELIMINATES deoxyATP; CATALYZES deoxyATP> deoxyAMP + PPi 11. REPEAT STEPS 8 – 10 WITH deoxyGTP, deoxyCTP, deoxyTTP, SEPARATELY. GO ON TO NEXT RESIDUE; AND ON AND ON: 25 MILLION SEQUENCING READS 400 BASES (AVERAGE)/READ 10 BILLION BASES = 10 GIGABASES

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