GENE TECHNOLOGY Chapter 13

Human Genome Sequencing of the human genome was completed by 2007 Create a map showing where genes are located on human chromosomes DNA sequencing has depended on advances in technology, starting with making recombinant DNA In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products

Fig. 20-1 Microarray An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes. This shows which genes are active in all cells. Figure 20.1 How can this array of spots be used to compare normal and cancerous tissues?

DNA Cloning and Its Applications Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids Plasmids are small circular extra-chromosomal DNA molecules that replicate separately (autonomously) from the bacterial chromosome. They are often used as vectors in genetic engineering. Cloned genes are useful for making copies of a particular gene and producing a protein product

Fig. 20-2a Transgenics Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome 2 2 Plasmid put into bacterial cell Figure 20.2 A preview of gene cloning and some uses of cloned genes Recombinant bacterium In gene cloning, the original plasmid is called a cloning vector A cloning vector is a DNA molecule that can carry foreign DNA into a host cell and replicate there

Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites A restriction enzyme usually makes many cuts, yielding restriction fragments DNA ligase is an enzyme that seals the bonds between restriction fragments

Recombinant bacterium Fig. 20-2b Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Protein harvested 4 Basic research and various applications Basic research on gene Basic research on protein Figure 20.2 A preview of gene cloning and some uses of cloned genes 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

Fig. 20-2 Cell containing gene of interest Bacterium 1 Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Being able to genetically engineer bacteria for crops help keep cost low. Growing non- virulent bacteria to clean oil spills is cost effective and a safer choice for our environment. 2 Plasmid put into bacterial cell Genetically engineered proteins could mean less invasive procedures. An alternative to surgery. Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Copies of gene Protein harvested Figure 20.2 A preview of gene cloning and some uses of cloned genes 4 Basic research and various applications 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

DNA cloning To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning DNA cloning yields multiple copies of a gene or other DNA segments.

Gene cloning involves using bacteria to make multiple copies of a gene Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA This results in the production of multiple copies of a single gene

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA know as VNTR’s (specific DNA code repeats unique to an individual). PCR and DNA Replication require the same ingredients to make a new strand (Helicase, DNA polymerase.) A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a Figure 20.8 The polymerase chain reaction (PCR)

Cycle 1 yields 2 molecules Fig. 20-8b 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension Figure 20.8 The polymerase chain reaction (PCR) New nucleo- tides

Cycle 2 yields 4 molecules Fig. 20-8c Cycle 2 yields 4 molecules Figure 20.8 The polymerase chain reaction (PCR)

molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Figure 20.8 The polymerase chain reaction (PCR)

molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8 TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleo- tides Figure 20.8 The polymerase chain reaction (PCR) Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

DNA Fingerprinting Used in criminal investigations: Detectives and forensic scientists use DNA information found at crime scene to understand what transpired and who committed the crime. Everyone’s DNA is unique due to their variable number tandem repeats.

Gel Electrophoresis – Another form of biotechnology Separates nucleic acids or proteins according to size (density) and charge.

Vaccines A vaccine is a substance used to promote the production of antibodies and provide immunity against the pathogen being introduced. Substances is either part of or completely made of a harmless version of the pathogen. DNA vaccines made using one or more genes from the pathogen. DNA vaccines do not have the capability of causing disease.