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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

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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

-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’


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


Vectors

Artificial chromosomes

-Used to clone very large DNA fragments

-Bacterial artificial chromosomes (BACs)

-Yeast artificial chromosomes (YACs)


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


DNA Libraries

Molecular hybridizationis 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


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

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


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


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


DNA Analysis


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 method was developed by Frederick Sanger

-Dideoxynucleotides are used as chain terminators in DNA synthesis reactions


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


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


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


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


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


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

-Baitvector: has DNA-binding domain

-Prey vector: has transcription-activating domain

-Neither of these alone can activate transcription


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


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

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


Agricultural Applications


Agricultural Applications


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


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


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


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


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


Agricultural Applications


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