Biotechnology Chapter 17

DNA Manipulation The molecular biology revolution started with the discovery of restriction endonucleases -Enzymes that cleave DNA at specific sites These enzymes are significant in two ways 1. Allow a form of physical mapping that was previously impossible 2. Allow the creation of recombinant DNA molecules (from two different sources)

DNA Manipulation Restriction enzymes recognize DNA sequences termed restriction sites There are two types of restriction enzymes: -Type I = Cut near the restriction site -Type II = Cut at the restriction site -The sites are palindromes -Both strands have same sequence when read 5’ to 3’

DNA Manipulation Type II enzymes produce staggered cuts that generate “sticky ends” -Overhanging complementary ends Therefore, fragments cut by the same enzyme can be paired DNA ligase can join the two fragments forming a stable DNA molecule

Gel Electrophoresis A technique used to separate DNA fragments by size; The gel (agarose or polyacrylamide) is subjected to an electrical field; The DNA, which is negatively-charged, migrates towards the positive pole -The larger the DNA fragment, the slower it will move through the gel matrix; DNA is visualized using fluorescent dyes

Transformation Transformation is the introduction of DNA from an outside source into a cell. Natural transformation occurs in many species -However, not in E. coli, which is used routinely in molecular biology labs -Artificial transformation techniques have been developed to introduce foreign DNA into it

Molecular Cloning A clone refers to a genetically identical copy; Molecular cloning is the isolation of a specific DNA sequence (usually protein-encoding) -Sometimes called gene cloning The most flexible and common host for cloning is E. coli Propagation of DNA in a host cell requires a vector

Vectors Plasmids are small, circular extrachromosomal DNA molecules -Used for cloning small pieces of DNA -Have three important components 1. Origin of replication 2. Selectable marker 3. Multiple cloning site (MCS)

Vectors

Vectors Phage vectors are modified bacterial viruses -Most based on phage lambda (l) of E. coli -Used to clone inserts up to 40 Kbp -Have two features not shared with plasmid vectors -They kill their host cells -They have linear genomes -Middle replaced with inserted DNA

Vectors

DNA Libraries A collection of DNA fragments from a specific source that has been inserted into host cells; A genomic library represents the entire genome; A cDNA library represents only the expressed part of the genome -Complementary DNA (cDNA) is synthesized from isolated mRNA using the enzyme reverse transcriptase

DNA Libraries Molecular hybridization is a technique used to identify specific DNAs in complex mixtures -A known single-stranded DNA or RNA is labeled -It is then used as a probe to identify its complement via specific base-pairing -Also termed annealing Molecular hybridization is the most common way of identifying a clone in a DNA library

DNA Analysis Restriction maps -Molecular biologists need maps to analyze and compare cloned DNAs; -The first maps were restriction maps -Initially, they were created by enzyme digestion & analysis of resulting patterns -Many are now generated by computer searches for cleavage sites

DNA Analysis Southern blotting -A sample DNA is digested by restriction enzymes & separated by gel electrophoresis; -Gel is transferred (“blotted”) onto a nitrocellulose filter -Then hybridized with a cloned, radioactively-labeled DNA probe -Complementary sequences are revealed by autoradiography

DNA Analysis DNA fingerprinting -An identification technique used to detect differences in the DNA of individuals; -Makes use of a variety of molecular procedures; -First used in a US criminal trial in 1987 -Tommie Lee Andrews was found guilty of rape

DNA Analysis DNA sequencing -A set of nested fragments is generated -End with known base -Separated by high-resolution gel electrophoresis, resulting in a “ladder” -Sequence is read from the bottom up

DNA Analysis DNA sequencing -The enzymatic technique develop by Frederick Sanger is powerful but is labor intensive and time-consuming -The development of automated techniques made sequencing faster and more practical -Fluorescent dyes are used instead of radioactive labels -Reaction is done in one tube -Data are assembled by a computer

DNA Analysis Polymerase chain reaction (PCR) -Developed by Kary Mullis -Allows the amplification of a small DNA fragment using primers that flank the region -Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) -Taq polymerase

DNA Analysis Polymerase chain reaction (PCR) -Has revolutionized science and medicine because it allows the investigation of minute samples of DNA -Forensics -Detection of genetic defects in embryos -Analysis of mitochondrial DNA from early human species

Genetic Engineering Has generated excitement and controversy. Expression vectors contain the sequences necessary to express inserted DNA in a specific cell type. Transgenic animals contain genes that have been inserted without the use of conventional breeding.

Genetic Engineering In vitro mutagenesis -Ability to create mutations at any site in a cloned gene -Has been used to produce knockout mice, in which a known gene is inactivated -The effect of loss of this function is then assessed on the entire organism -An example of reverse genetics

Medical Applications Human proteins -Medically important proteins can be produced in bacteria -Human insulin -Interferon -Atrial peptides -Tissue plasminogen activator -Human growth hormone

Medical Applications

Medical Applications Vaccines -Subunit vaccines: Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome -DNA vaccines: Depend on the cellular immune response (not antibodies)

Medical Applications

Medical Applications Gene therapy -Adding a functional copy of a gene to correct a hereditary disorder -Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems -Successful at first, but then patients developed a rare leukemia

Agricultural Applications Herbicide resistance -Broadleaf plants have been engineered to be resistant to the herbicide glyphosate -This allows for no-till planting

Agricultural Applications Pest resistance -Insecticidal proteins have been transferred into crop plants to make them pest-resistant -Bt toxin from Bacillus thuringiensis Golden rice -Rice that has been genetically modified to produce b-carotene (provitamin A) -Converted in the body to vitamin A

Agricultural Applications Daffodil phytoene synthase gene (psy) psy crtI lcy Phytoene Carotene desaturase -Cyclase Genes introduced into rice genome Expression in endosperm GGPP Lycopene -Carotene (Provitamin A) Bacterial carotene gene (crtI) lycopene -cyclase gene (lcy) Rice chromosome

Agricultural Applications