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. Fig. 20.3 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5353 3535 1 One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2

3. Fig. 20.2 DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA ( plasmid ) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Plasmid Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 2 4 1 3

4. Fig. 20.6 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail DNA strand Primer mRNA Degraded mRNA DNA polymerase cDNA

5. Fig. 20.9a Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE 1 2 Power source – + + –

6. Fig. 20.8 5 Genomic DNA TECHNIQUE Cycle 1 yields 2 molecules Denaturation Annealing Extension Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Target sequence Primers New nucleo- tides 3 3 3 3 5 5 5 1 2 3

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

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

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

11. Fig. 20.22 Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient. 1 2 3 4

12. Fig. 20.25 Site where restriction enzyme cuts T DNA Plant with new trait Ti plasmid Agrobacterium tumefaciens DNA with the gene of interest Recombinant Ti plasmid TECHNIQUE RESULTS