What are the Techniques of Biotechnology ?

1. What are the Techniques of Biotechnology ? • Restriction Endonucleases: enzymes that cut DNA at specific codes (nucleotide sequences) • Can buy from suppliers: ex. cut at ATATAT • DNA Fingerprinting: sequence code of sample DNA (a portion of genome) or compare digested samples via gel electrophoresis • In crime and paternity testing, evidence sample compared to suspects’ samples; if different codes, or different patterns on gel, then cannot be “donor”; if match, % likelihood based on size of genome sequenced, or frequency of gel pattern in population • Rape charges even filed against “unknown person” with sample DNA (statute of limitations was approaching) • DNA very strong evidence for innocence, not as strong for guilt; but has been used as primary evidence in capital cases (resulted in executions) • DNA from whale-meat in Japanese restaurants showed many whale and dolphin species sold despite moratorium on most species • Polymerase chain reaction: machine that replicates a small sample of DNA into a larger amount of identical sample (enough to work with)

2. Restriction site 5 3 3 5 DNA Restriction enzymecuts sugar-phosphatebackbones. 1 Fig. 20.3 Sticky end DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. 2 One possible combination DNA ligaseseals strands. 3 Recombinant DNA molecule

3. Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Fig. 20.2 Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

4. DNA innucleus mRNAs in cytoplasm Reversetranscriptase Poly-A tail Fig. 20.6 mRNA Primer DNAstrand DegradedmRNA DNA polymerase cDNA

5. TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes Anode Cathode – + Fig. 20.9a Gel 1 Powersource – + Longermolecules 2 Shortermolecules

6. 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Fig. 20.8 Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

7. What are the Applications of Biotechnology? • Human Genome Project: highly collaborative; completed in early 2001; human genome mapped and sequenced; next step is understanding the functions of genes (and resulting proteins) • Reasoning was that several genetic diseases would become better under- stood during the project (sooner than if each was studied independently) • Genetic Screening: geneticists use interviews and DNA fingerprinting; concerns regarding insurance and potential discrimination • Genetic Therapy: inject “healthy genes” into blood; some success in diseases of the blood (immune disorders) • Genetic Engineering (recombinant technology): manipulate genes in fertilized egg; replace un-wanted gene with copy of desired gene • Transgenic Organisms: because genetic code and ribosome “machinery” shared in all organisms, bacteria (and other organisms) can make human proteins if appropriate gene is inserted into cell (or fertilized egg); such proteins are often medicines (ex., replace casein gene in milk of sheep or goats with desired gene) • Agricultural Applications: genes for natural insecticides, drought-resistance, and frost-resistance transferred to crops

8. Chromosome bands Cytogenetic map Genes located by FISH Fig. 21.2 Linkage mapping 1 Genetic markers Physical mapping 2 Overlapping fragments DNA sequencing 3

9. 1 Cut the DNA into overlapping fragments short enough for sequencing 2 Clone the fragments in plasmid or phage vectors. Fig. 21.3 3 Sequence each fragment. 4 Order the sequences into one overall sequence with computer software.

10. Normal -globin allele Normalallele Sickle-cellallele Fig. 20.10 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

11. 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

12. TECHNIQUE Agrobacterium tumefaciens Tiplasmid Fig. 20.25 Site whererestrictionenzyme cuts T DNA RESULTS DNA withthe geneof interest RecombinantTi plasmid Plant with new trait