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Explore the process of using biotechnology to produce human insulin using recombinant DNA technology and genetic engineering techniques. Learn about the role of restriction enzymes, plasmids, and gene insertion in creating recombinant bacteria. Discover methods for identifying and detecting the gene of interest, and explore potential applications in medical research and treatment.
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Biotechnology • Making microbes work for you! • Base pairing between nucleic acids is really useful What if we wanted a lot of human insulin?
Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Fig. 20-2a Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 2 Plasmid put intobacterial cell Recombinantbacterium
Restriction enzymes: • Enzymes that break the sugar-phosphate backbone of DNA (“restrict” the DNA) at specific sequences • Many leave short, single-stranded “sticky ends” after they cut DNA Table of restriction enzymes
Fig. 20-3-1 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end
Fig. 20-3-2 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination
Fig. 20-3-3 Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule
Fig. 20-4-1 Hummingbird cell TECHNIQUE Bacterial cell Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments
Fig. 20-4-2 Hummingbird cell TECHNIQUE Bacterial cell Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids
Fig. 20-4-3 Hummingbird cell TECHNIQUE Bacterial cell Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids
Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS One of manybacterial clones
Fig. 20-4-4 Hummingbird cell TECHNIQUE Bacterial cell Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids How do we know which plasmid has our gene of interest (i.e. insulin)? RESULTS One of manybacterial clones
• Base pairing (“hybridization”) between nucleic acids is very powerful TECHNIQUE Radioactivelylabeled probemolecules ProbeDNA Gene ofinterest Fig. 20-7 Multiwell platesholding library clones Single-strandedDNA from cell Film Nylon membrane Nylonmembrane Location ofDNA with thecomplementarysequence
pET11c: an example of a plasmid Will E. coli be able to express human insulin?
Fig. 20-6-5 DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail mRNA Reverse transcriptase allows RNA to be copied (“reverse transcribed”) into cDNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA
What if we wanted to introduce a functional insulin gene into a patient?
Not yet feasible Clonedgene Insert RNA version of normal alleleinto retrovirus. 1 Viral RNA Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured. 2 Fig. 20-22 Retroviruscapsid Viral DNA carrying the normalallele inserts into chromosome. 3 Bonemarrowcell frompatient Bonemarrow Inject engineeredcells into patient. 4
TECHNIQUE Heavyweight Restrictionfragments I II III Nitrocellulosemembrane (blot) DNA + restriction enzyme Gel Sponge I Normal-globinallele II Sickle-cellallele III Heterozygote Papertowels Fig. 20-11 Alkalinesolution 2 1 3 Preparation of restriction fragments DNA transfer (blotting) Gel electrophoresis Radioactively labeledprobe for -globin gene Probe base-pairswith fragments I II III I II III Fragment fromsickle-cell-globin allele Film overblot Fragment fromnormal -globin allele Nitrocellulose blot 4 5 Probe detection Hybridization with radioactive probe
TECHNIQUE 1 cDNA synthesis mRNAs cDNAs Fig. 20-13 Primers 2 PCR amplification -globingene 3 Gel electrophoresis Embryonic stages RESULTS 1 2 3 4 5 6
Fig. 20-14 50 µm
TECHNIQUE Tissue sample 1 Isolate mRNA. 2 Make cDNA by reversetranscription, usingfluorescently labelednucleotides. mRNA molecules Fig. 20-15 Labeled cDNA molecules(single strands) DNA fragmentsrepresentingspecific genes 3 Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray. DNA microarray DNA microarraywith 2,400human genes 4 Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.
