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Biotechnology Chapter 17.

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Presentation on theme: "Biotechnology Chapter 17."— Presentation transcript:

1 Biotechnology Chapter 17

2 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)

3 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’

4 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


6 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


8 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

9 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

10 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)

11 Vectors

12 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

13 Vectors

14 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


16 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

17 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

18 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


20 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

21 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

22 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

23 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


25 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

26 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.

27 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


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

30 Medical Applications

31 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)

32 Medical Applications

33 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

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

35 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

36 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

37 Agricultural Applications

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