BiotechnologyChapter 17

DNA Manipulation

• Restriction endonucleases revolutionized molecular biology

• Enzymes that cleave DNA at specific sites– Used by bacteria against viruses

• Restriction enzymes significant– Allow a form of physical mapping that was

previously impossible– Allow the creation of recombinant DNA

molecules (from two different sources) 2

• 3 types of restriction enzymes

• Type I and III cleave with less precision and are not used in manipulating DNA

• Type II – Recognize specific DNA sequences– Cleave at specific site within sequence– Can lead to “sticky ends” that can be joined

• Blunt ends can also be joined

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• DNA ligase– Joins the two fragments forming a stable DNA

molecule– Catalyzes formation of a phosphodiester bond

between adjacent phosphate and hydroxyl groups of DNA nucleotides

– Same enzyme joins Okazaki fragments on lagging strand in replication

Gel Electrophoresis

• Separate DNA fragments by size• Gel made of agarose or polyacrylamide• Submersed in buffer that can carry current• Subjected to an electrical field• Negatively-charged DNA migrates towards the

positive pole• Larger fragments move slower, smaller move

faster• DNA is visualized using fluorescent dyes

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Transformation

• Introduction of DNA from an outside source into a cell

• Natural process in many species– E. coli does not

• Temperature shifts can induce artificial transformation in E. coli

• Transgenic organisms are all or part transformed cells

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Molecular Cloning

• Clone – genetically identical copy

• Molecular cloning – isolation of a specific DNA sequence (usually protein-encoding)– Sometimes called gene cloning

• The most flexible and common host for cloning is E. coli – Vector – carries DNA in host and can

replicate in the host– Each host–vector system has particular uses

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Vectors

• Plasmids – Small, circular chromosomes– Used for cloning small pieces of DNA– 3 components

• Origin of replication – allows independent replication

• Selectable marker – allows presence of plasmid to be easily identified

• Multiple cloning site (MCS)

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

• This process involves three steps:1. Plating the library

• Physically the library is a collection of bacteria or viruses in bacteria

2. Replicating the library

3. Screening the library• Probe is specific sequence of interest

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DNA Analysis

• Restriction maps– Molecular biologists need maps to analyze

and compare cloned DNAs– Initially, created by enzyme digestion,

separation by electrophoresis, and analysis of resulting patterns

– Many are now generated by computer searches for cleavage sites

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• Southern blotting– Sample DNA is digested by restriction enzymes

and separated by gel electrophoresis– Double-stranded DNA denatured into single-

strands– Gel “blotted” with filter paper to transfer DNA– Filter is incubated with a labeled probe

consisting of purified, single-stranded DNA corresponding to a specific gene

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• Northern blotting– mRNA is separated by electrophoresis and

then blotted onto the filter

• Western blotting– Proteins are separated by electrophoresis and

then blotted onto the filter– Detection requires an antibody that can bind

to one protein

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• RFLP analysis– Restriction fragment length polymorphisms– Generated by point mutations or sequence

duplications– Restriction enzyme fragments are often not

identical in different individuals– Can be detected by Southern blotting

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• DNA fingerprinting– Identification technique used to detect

differences in the DNA of individuals– Population is polymorphic for these markers– Using several probes, probability of identity

can be calculated or identity can be ruled out– First used in a U.S. criminal trial in 1987

• Tommie Lee Andrews was found guilty of rape

– Also used to identify remains

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DNA Analysis

• Polymerase chain reaction (PCR)– Developed by Kary Mullis

• Awarded Nobel Prize

– 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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• Applications of PCR– Allows the investigation of minute samples of

DNA– Forensics – drop of blood, cells at base of a

hair– Detection of genetic defects in embryos by

analyzing a single cell– Analysis of mitochondrial DNA from early

human species

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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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• In vitro mutagenesis– Ability to create mutations at any site in a

cloned gene– Has been used to produce knockout mice

• 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

• Medically important proteins can be produced in bacteria– Human insulin– Interferon– Atrial peptides– Tissue plasminogen activator – Human growth hormone– Problem has been purification of desired

proteins from other bacterial proteins31