n Understanding and Manipulating Genomes n One of the greatest achievements of modern science –Has been the sequencing of the human genome, which was largely completed by 2003 n DNA sequencing accomplishments –Have all depended on advances in DNA technology, starting with the invention of methods for making recombinant DNA Chapter 20 & 21~Biotechnology & Genomics

n DNA technology has launched a revolution in the area of biotechnology –The manipulation of organisms or their genetic components to make useful products n Variation: Differences within members of a species. Golden Rice, A GMO containing Vitamin A

Using Genetics Technology Before Recombinant DNA Technology n Selective Breeding: Choosing organisms that breed together with the hopes of producing “desirable traits” in the next generation. –Hybrid: mix between two distinct things. –Inbreeding: mating between individuals with similar traits. It's pretty much my favorite animal. It's like a lion and a tiger mixed... bred for its skills in magic. -Napoleon Dynamite (in reference to ligers)

Recombinant DNA n DNA in which genes from 2 different sources are linked –Genetic engineering: direct manipulation of genes for practical purposes

n DNA cloning permits production of multiple copies of a specific gene or other DNA segment n To work directly with specific genes –Scientists have developed methods for preparing well-defined, gene-sized pieces of DNA in multiple identical copies, a process called gene cloning

DNA Cloning and Its Applications: A Preview n Most methods for cloning pieces of DNA in the laboratory –Share certain general features, such as the use of bacteria and their plasmids

Bacterium Bacterial chromosome Plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest DNA of chromosome Recombinate bacterium Protein harvested Basic research on protein Gene of interest Copies of gene 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 Protein expressed by gene of interest 3 Gene inserted into plasmid 1 Plasmid put into bacterial cell 2 Host cell grown in culture, to form a clone of cells containing the “cloned” gene of interest 3 Basic research and various applications 4

20.1: Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes –Cut DNA molecules at a limited number of specific DNA sequences, called restriction sites Restriction Enzyme Animation

A restriction enzyme will usually make many cuts in a DNA molecule –Yielding a set of restriction fragments The most useful restriction enzymes cut DNA in a staggered way –Producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments DNA ligase is an enzyme –That seals the bonds between restriction fragments

Restriction site DNA G A A T T C C T T A A G Sticky end Fragment from different DNA molecule cut by the same restriction enzyme One possible combination Recombinant DNA molecule G C T T A A A A T T C G C T T A A G G G G A A T TCA A T T C C T T A A G C T T A A G Using a restriction enzyme and DNA ligase to make recombinant DNA Restriction enzyme cuts the sugar phosphate backbones at each arrow 1 DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. 2 DNA ligase seals the strands. 3

Cloning a Eukraryotic Gene in a Bacterial Plasmid In gene cloning, the original plasmid is called a cloning vector –Defined as a DNA molecule that can carry foreign DNA into a cell and replicate there Animation

Producing Clones of Cells 1 Isolate plasmid DNA and human DNA. 2 Cut both DNA samples with the same restriction enzyme 3 Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. APPLICATION Cloning is used to prepare many copies of a gene of interest for use in sequencing the gene, in producing its encoded protein, in gene therapy, or in basic research. TECHNIQUE In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the amp R gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes  -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples. Human DNA fragments Human cell Gene of interest Bacterial cell amp R gene Bacterial plasmid Restriction site Recombinant DNA plasmids lacZ gene

RESULTS Only a cell that took up a plasmid, which has the amp R gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X- gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe researchers can identify clones of bacterial cells carrying the gene of interest. Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone Colony carrying recombinant plasmid with disrupted lacZ gene Recombinant bacteria 4 Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. 5 Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow.

Making HGH using bacteria

Steps for eukaryotic gene cloning (Review) n Isolation of cloning vector (bacterial plasmid) & gene- source DNA (gene of interest) n Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase n Introduction of cloning vector into cells (transformation by bacterial cells) n Cloning of cells (and foreign genes) n Identification of cell clones carrying the gene of interest

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) n The polymerase chain reaction, PCR –Can produce many copies of a specific target segment of DNA –Uses primers that bracket the desired sequence –Uses a heat-resistant DNA polymerase

Polymerase chain reaction (PCR) n Copies of any piece of DNA without the use of the body (in vitro) n Primers: short sequences of complimentary DNA to start polymerization. n Comprehensive website for 20.1 Comprehensive website for 20.1 Comprehensive website for 20.1

20.2: DNA Analysis n Restriction fragment analysis detects DNA differences that affect restriction sites n Restriction fragment analysis –Can rapidly provide useful comparative information about DNA sequences

DNA Analysis n Gel electrophoresis: separates nucleic acids or proteins on the basis of size or electrical charge creating DNA bands of varying length

20.3 – 20.4: Practical DNA Technology Uses n Diagnosis of disease n Human gene therapy n Pharmaceutical products (vaccines) n Forensics n Animal husbandry (transgenic organisms) n Genetic engineering in plants n Ethical concerns?

Genetic Engineering in Medicine and Society n Very useful for medicine since many disorders are caused when the body fails to make proteins. –Gene therapy: putting a healthy copy of a gene into the cells of a person with a bad copy –Stem Cell Research: use of unspecialized cells for replacement of “defective” cells n DNA fingerprints: used to identify an organism n DNA Fingerprinting Activity DNA Fingerprinting Activity DNA Fingerprinting Activity n Using the Evidence Activity Using the Evidence Activity Using the Evidence Activity

Fig Cultured stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) Different culture conditions Different types of differentiated cells Blood cells Nerve cells Liver cells Cells generating all embryonic cell types Adult stem cells Cells generating some cell types Embryonic stem cells From bone marrow in this example

Genetic Engineering in Agriculture n Make plants more drought resistant, speed up ripening, improve nutritional value, and insect resistant. n Transgenic animals: animals with foreign DNA that can do special things.

21.1: Genomics n Entire genomes can be mapped at the DNA level n The Human Genome Project –Sequenced the human genome n Scientists have also sequenced genomes of other organisms –Providing important insights of general biological significance

DNA Sequencing n Determination of nucleotide sequences n Genomics: the study of genomes based on DNA sequences n Human Genome Project n Activity Activity