Biotechnology Chapter 17. 2 DNA Manipulation The molecular biology revolution started with the discovery of restriction endonucleases -Enzymes that cleave.

BiotechnologyChapter 17

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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)

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DNA Manipulation

Restriction enzymes recognize DNA sequences termed restriction sites

There are two types of restriction enzymes:

-Type I = Cut near the restriction site

-Rarely used in DNA manipulation

-Type II = Cut at the restriction site

-The sites are palindromes

-Both strands have same sequence when read 5’ to 3’

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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

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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

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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

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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

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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)

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Vectors

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Vectors

Phage vectors are modified bacterial viruses

-Most based on phage lambda () 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

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Vectors

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Vectors

Artificial chromosomes

-Used to clone very large DNA fragments

-Bacterial artificial chromosomes (BACs)

-Yeast artificial chromosomes (YACs)

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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

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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

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DNA Libraries

Molecular hybridization is the most common way of identifying a clone in a DNA library

-This process involves three steps:

1. Plating the library

2. Replicating the library

3. Screening the library

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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

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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

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DNA Analysis

Northern blotting

-mRNA is electrophoresed and then blotted onto the filter

Western blotting

-Proteins are electrophoresed and then blotted onto the filter

-Detection requires an antibody that can bind to one protein

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DNA Analysis

RFLP analysis

-Restriction fragment length polymorphisms (RFLPs) are generated by point mutations or sequence duplications

-These fragments are often not identical in different individuals

-Can be detected by Southern blotting

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DNA Analysis

DNA fingerprinting

-An identification technique used to detect differences in the DNA of individuals

-Makes use of a variety of molecular procedures, including RFLP analysis

-First used in a US criminal trial in 1987

-Tommie Lee Andrews was found guilty of rape

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DNA Analysis

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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

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DNA Analysis

DNA sequencing

-The enzymatic method was developed by Frederick Sanger

-Dideoxynucleotides are used as chain terminators in DNA synthesis reactions

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DNA Analysis

DNA sequencing-The enzymatic technique 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

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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

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After 20 cycles, a single fragment produces over one million (220) copies!

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After 20 cycles, a single fragment produces over one million (220) copies!(Cont.)

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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

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DNA Analysis

Yeast two-hybrid system-Used to study protein-protein interactions-Gal4 is a transcriptional activator with a modular structure-The Gal4 gene is split into two vectors

-Bait vector: has DNA-binding domain -Prey vector: has transcription-

activating domain -Neither of these alone can

activate transcription

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DNA Analysis

Yeast two-hybrid system

-When other genes are inserted into these vectors, they produce fusion proteins

-Contain part of Gal4 and the protein of interest

-If the proteins being tested interact, Gal4 function will be restored

-A reporter gene will be expressed

-Detected by an enzyme assay

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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

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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

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Medical Applications

Human proteins

-Medically important proteins can be produced in bacteria

-Human insulin

-Interferon

-Atrial peptides

-Tissue plasminogen activator

-Human growth hormone

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Medical Applications

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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)

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Medical Applications

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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

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Agricultural Applications

Ti (tumor-inducing) plasmid is the most used vector for plant genetic engineering

-Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants

-However, bacterium does not infect cereals such as corn, rice and wheat

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Agricultural Applications

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Agricultural Applications

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Agricultural Applications

Gene guns

-Uses bombardment with tiny gold particles coated with DNA

-Possible for any species

-However, the copy number of inserted genes cannot be controlled

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Agricultural Applications

Herbicide resistance

-Broadleaf plants have been engineered to be resistant to the herbicide glyphosate

-This allows for no-till planting

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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 -carotene (provitamin A)

-Converted in the body to vitamin A

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Agricultural Applications

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Agricultural Applications

Adoption of genetically modified (GM) crops has been resisted in some areas because of questions about:

-Crop safety for human consumption

-Movement of genes into wild relatives

-Loss of biodiversity

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Agricultural Applications

Biopharming

-Transgenic plants are used to produce pharmaceuticals

-Human serum albumin

-Recombinant subunit vaccines

-Against Norwalk and rabies viruses

-Recombinant monoclonal antibodies

-Against tooth decay-causing bacteria

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Agricultural Applications

Transgenic animal technology has not been as successful as that in plants

-One interesting example is the EnviroPig

-Engineered to carry the gene for the enzyme phytase

-Breaks down phosphorus in feed

-Reduces excretion of harmful phosphates in the environment

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Agricultural Applications