Avery, MacLeod, and McCarty 1944 Used bacteria from Griffith’s mouse experiment Denatured proteins in membrane and discovered that the DNA still could make other bacteria pathogenic

Biotechnology – pg. 140 in Cliffs Ch. 20 in text  Recombinant DNA – a combination of DNA segments from two different sources  Can occur through transduction, conjugation, transformation  Can also occur during crossing over during meiosis in eukaryotes  Biotechnology – use of biological systems to produce products like medicine  Often use bacteria and viruses in experiments and production of products

Recombinant DNA technology  Set of lab techniques for combining genes from different sources.  Requires the “cutting” of DNA using restriction enzymes  Restriction enzymes cut DNA at very specific sequences called restriction sites  Using bacterial plasmids we can clone specific genes to produce proteins of interest  Ex. Medicine, farming, oil clean up

Creating Sticky Ends

© 2011 Pearson Education, Inc. Animation: Restriction Enzymes Right-click slide / select “Play”

Using Restriction Enzymes to cut DNA Restriction Enzyme Video

Figure Recombinant DNA molecule One possible combination DNA ligase seals strands DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA Sticky end GAATTC CTTAAG CTTAA G AATTC G G G CTTAA G G G G AATT C C TTAA

Recombinant DNA

Figure DNA in nucleus mRNAs in cytoplasm mRNA Reverse transcriptase Poly-A tail DNA strand Primer DNA polymerase cDNA A A A T T T T T

Figure 20.2 Bacterium Bacterial chromosome Plasmid 2134 Gene inserted into plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed from gene of interest Protein harvested Copies of gene Basic research and various applications Basic research on protein Basic research on gene 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 hormone treats stunted growth

DNA cloning 1.Use restriction enzyme to cut a sample of DNA in test tube – this will create fragments with sticky ends, some will have our gene of interest 2.Cut a plasmid (cloning vector) with one restriction site for the restriction enzyme – the plasmid will now have the same sticky ends (plasmid should also be resistant to antibiotic like ampicillin) 3.Mix the foreign DNA with the plasmids 4.Apply DNA ligase

Transformation Time  Place the engineered plasmid into bacterial culture (in test tube)  Heat shock and let transformation occur  Plate the bacteria and those that grow on ampicillin will have “transformed” with the foreign gene of interest

Genomic Library  At the end, the bacteria now contain our gene of interest – genomic library  Now the gene can be transcribed and translated to make the protein of interest  This DNA is without introns because it was made from mRNA using reverse transcriptase before the experiment. cDNA

Figure 20.4 Bacterial plasmid TECHNIQUE RESULTS amp R gene lacZ gene Restriction site Hummingbird cell Sticky ends Gene of interest Humming- bird DNA fragments Recombinant plasmidsNonrecombinant plasmid Bacteria carrying plasmids Colony carrying non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene One of many bacterial clones

Figure 20.5 Foreign genome Cut with restriction enzymes into either small fragments large fragments or Recombinant plasmids Plasmid clone (a) Plasmid library (b) BAC clone Bacterial artificial chromosome (BAC) Large insert with many genes (c) Storing genome libraries

Storing Cloned Genes in DNA Libraries A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome A genomic library that is made using bacteriophages is stored as a collection of phage clones A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene This process is called nucleic acid hybridization © 2011 Pearson Education, Inc.

A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is – Then we would synthesize this probe © 2011 Pearson Education, Inc. 5 3  CTCAT CACCGGC  5 3 G A G T A G T G G C C GG A G T A G T G G C C G

Figure 20.7 Radioactively labeled probe molecules Gene of interest Probe DNA Single- stranded DNA from cell Film Location of DNA with the complementary sequence Nylon membrane Multiwell plates holding library clones TECHNIQUE GAGTAGTGGCCG  CTCATCACCGGC 

Finding specific mutations Gel Electrophoresis In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition Genetic markers called SNPs (single nucleotide polymorphisms) occur on average every 100–300 base pairs © 2011 Pearson Education, Inc.

Figure DNA SNP Normal allele Disease-causing allele T C

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

Figure DNA  restriction enzyme TECHNIQUE I Normal  -globin allele II Sickle-cell allele III Heterozygote Restriction fragments Nitrocellulose membrane (blot) Heavy weight Gel Sponge Alkaline solution Paper towels II IIII II IIII II IIII Preparation of restriction fragments Gel electrophoresis DNA transfer (blotting) Radioactively labeled probe for  -globin gene Nitrocellulose blot Probe base-pairs with fragments Fragment from sickle-cell  -globin allele Fragment from normal  - globin allele Film over blot Hybridization with labeled probe Probe detection 5

Gel Box

Applications of Gene Technology  DNA Fingerprint

DNA Fingerprinting DNA fingerprinting

Making copies of DNA - PCR  Polymerase chain reaction (PCR) makes copies of DNA in order to have enough sample to run many tests on.  You take the sample of DNA, and heat them along with DNA polymerase and A,T,C,G “primers”  They will make millions of copies of the sample.

Figure 20.8 Genomic DNA Target sequence Denaturation Annealing Extension Primers New nucleotides Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence TECHNIQUE

Studying the Expression of Interacting Groups of Genes  Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays  DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions © 2011 Pearson Education, Inc.

Isolate mRNA TECHNIQUE Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed in the tissue sample. Tissue sample mRNA molecules Labeled cDNA molecules (single strands) DNA fragments representing a specific gene DNA microarray DNA microarray with 2,400 human genes Figure 20.15

Using reverse transcriptase in gene therapy  Isolate mRNA and use an enzyme called reverse transcriptase to create DNA  These artificial DNA molecules can be inserted via a virus into a patient’s cells, then into the patient.

Gene Therapy in humans

Gene technology in Farming

Golden Rice  Rice injected with DNA that codes for beta- carotene that we use to make vitamin A

DNA injection

Cloning Plants: Single-Cell Cultures One experimental approach is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism Plant cloning is used extensively in agriculture © 2011 Pearson Education, Inc.

Figure Cross section of carrot root 2-mg fragments Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. Adult plant

Reproductive Cloning of Mammals In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus © 2011 Pearson Education, Inc.

Figure Mammary cell donor TECHNIQUE RESULTS Cultured mammary cells Egg cell from ovary Egg cell donor Nucleus removed Cells fused Grown in culture Implanted in uterus of a third sheep Embryonic development Nucleus from mammary cell Early embryo Surrogate mother Lamb (“Dolly”) genetically identical to mammary cell donor

DNA Sequencing Relatively short DNA fragments can be sequenced by the dideoxy chain termination method, the first automated method to be employed Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment The DNA sequence can be read from the resulting spectrogram © 2011 Pearson Education, Inc.

Figure DNA (template strand) TECHNIQUE 5 3 C C C C T T T G G A A A A G T T T DNA polymerase Primer 5 3 P P P OH G dATP dCTP dTTP dGTP Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) P P P H G ddATP ddCTP ddTTP ddGTP 5 3 C C C C T T T G G A A A A DNA (template strand) Labeled strands ShortestLongest 5 3 ddC ddG ddA ddG ddT ddC G T T T G T T T C G T T T C T T G G T T T C T G A G T T T C T G A A G T T T C T G A A G G T T T C T G A A G T G T T T C T G A A G T C G T T T C T G A A G T C A Direction of movement of strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last nucleotide of longest labeled strand Last nucleotide of shortest labeled strand G G G A A A C C T

Gene Sequencing Sanger Method of Sequencing DNA sequencing machine ad