Chapter 8• Recombinant DNA• Genetic Engineering• Genetic manipulation

Donor organism VectorDNA

DNA fragments

RecombinantDNA

Generating Recombinant DNA Molecules

Plasmids are extrachromosomal self-replicating DNA molecules

Making recombinant DNA

Techniques required: - DNA isolation- DNA fragmentation (cutting)- DNA ligation (joining)- amplification of recombinant DNA

DNA isolation

Plasmid DNA can be isolated by centrifugation in a CsCl gradient in the presence ofethidium bromide that intercalates into DNA (more into linear DNA). Modern methodsinvolve chromatographic steps that are simple and quick.

Donor DNA– genomic DNA from chromosomes

(usually too large to clone directly into plasmids)– cDNA (complementary DNA) derived by action of

reverse transcriptase from (usually) mRNA template– chemically synthesized oligonucleotides

Isolation of mRNA (polyA RNA)

mRNA is only about 1% of total cellular RNA.Eukaryotic mRNA is purified by making useof the poly(A) tails.

Making a cDNA library

• Isolation of mRNA (polyA RNA)• Preparation of cDNA

Preparation of cDNA

The enzyme reverse transcriptase(originally isolated from retro viruses)polymerizes ssDNA by using ssRNA astemplate (step 2). For the other steps ofthe procedure various other enzymes areused ( DNA polymerase, ligase, methylase).cDNAs are often cloned with the help ofphage vectors, because these are veryefficient for small DANN quantities (seelater)

Cutting DNA by restriction enzymes

Restriction enzymes (are endonucleases) cut DNA molecules at specificsequences

Restriction enzymes are endonucleases that protect bacteria against foreign DNA. Inthe bacterial DNA the recognition sequences are methylated. This prevents cutting ofthe bacterial DNA by endogenous restriction enzymes.

Some examples for restriction enzymes.

Note that restriction enzymes can produce cutswith overhanging ends (sticky ends) or withblunt ends. There are two types of sticky ends,one with 5' overhangs and one with 3'overhangs. The recognition sequence ofrestriction enzymes is a palindrome.

Joining of DNA fragments byDNA ligase.

Restriction fragments withcomplementary “sticky ends” areligated more easily.

Generating a recombinantDNA plasmid containinggenes from donor DNA.

Replicating Recombinant DNA

• Polymerase Chain Reaction (PCR)(Selective replication in a test tube)Kary Banks Mullis, Nobelpreis 1993

• Cloning Recombinant DNA(Selective replication in a host cell)

Polymerase chain reaction

Amplifying recombinant DNA

Transformed bacterial cells cangrow in the presence of aselectable agent, because thevector contains a selectablemarker, for example anantibiotic resistance gene.

cell multiplication

Amplifying recombinant DNAby cloning.

DNA cloning with bacterial vectors

• Recombinant DNA technology depends on the ability toproduce large numbers of identical DNA molecules (clones)

• Clones are typically generated by placing a DNA fragment ofinterest into a vector DNA molecule, which can replicate in ahost cell

• When a single vector containing a single DNA fragment isintroduced into a host cell, large numbers of the fragmentare reproduced along with the vector

• Two common vectors are E. coli plasmid vectors andbacteriophage l vectors

Plasmid vectors

Advanced vectors contain in addition to aselectable marker (drug resistance gene)a marker for visible selection of insertDNA. The polylinker or multible cloningsite contains unique restriction sites.These vectors are present in high copynumbers in the bacterial cells.

blue white

Plasmid vectors for very large DNA inserts

So-called BAC (bacterial artificial chromosomes) are derived fromF-plasmids. DNA fragments up to 150 kb can be cloned into thesevectors that are used to clone genomic DNA (genome projects).These vectors are present in low copy numbers in the bacterialcells.

Phage lambda as cloning vector for making a DNA library

Definition of a genomic or cDNA library?

Constructing DNA libraries with λ phage and other cloning vectors

• Cloning all of the genomic DNA of higher organisms into plasmidvectors is not practical due to the relatively low transformationefficiency of E. coli and the small number of transformed coloniesthat can be grown on a typical culture plate

• Cloning vectors derived from bacteriophage λ do not suffer fromsuch limitations

• A collection of clones that includes all the DNA sequences of a givenspecies is called a genomic library

• A genomic library can be screened for clones containing a sequenceof interest

Part of the phage genome is not essential for phage propagationand can be replaced by foreign DNA (about 20 kb).

Assembly of lambda phages

Lambda heads are filled with DNA fromlong linear multimeric (concatemers) donorDNA. Two COS sites define one unit of forfilling into the heads. Packaging is worksmost efficiently if the distance between twoCOS sites is about 50 kb.

For cloning, recominant lambda units areproduced that contain about 20 kb offoreign DNA. Long concatemers are createdthat can be assembled into phage particlesin vitro.

A genomic library should completely represent the DNA of an organism. Thereforethe genomic DNA is digested with a frequently cutting restriction enzyme, however,under conditions that produce long DNA fragments.

partial DNA digest

The lambda cloning procedure A)

The lambda cloning procedure B)

Delivery of recombinant DNA into bacterial cells

Finding specific clones (genes)

• by using nucleic acid probes • by using antibodies• by functional complementation

Finding specific clones (genes) by using nucleicacid probes.

A clone that contains a specific (known) DNAsequence can be identified in a library byhybridization with that specific DNA fragment.For example a genomic clone can be identifiedby using the corresponding cDNA.

Identifying DNA molecules

• It is often a challenge to identify desired gene in library ofthousands of clones

• Using nucleotide probes– principle based on base-pair complementarity– colonies or phage plaques are transferred to

membrane, lysed and DNA is denatured– probe is applied to membrane

• labeled with radioactive isotope or fluorescent dye• probe forms double helix with complementary DNA

– hybrid DNA is identified in autoradiogram or byexposure to exciting wavelength of light

Nucleotide probes

• Multiple possible sources– previously cloned genomic DNA or cDNA from another

species, tissue, etc.– PCR-amplified DNA– synthetic oligonucleotide

• reverse translated from amino acid sequence, ifknown

• synthesized by machine– RNA, such as rRNA or tRNA

• Hybridization of probe to complement is sensitive totemperature and salt

Finding specific clones (genes) by using nucleic acid probes.

A amino acid sequence can be used in order to generate oligonucleotides that match thecorresponding coding sequence of a gene. In this way, the gene encoding a knownprotein can be isolated.

Finding specific clones (genes) by functionalcomplementation.

A mutation in bacteria or in a fungus can be complemented bytransformation with cloned genes from a library. Complementationidentifies a gene with similar function. With this technique, forexample transporters for sugars or amino acids have beenidentified.

Cloning a gene by tagging (insertion mutagenesis)

The insertion of a known piece ofDNA (= tag, for example T-DNA,transposable elements) into agene leads to mutation of thatgene. The inserted DNA is thenused as tag to clone the gene.

For this procedure no knowledgeabout the gene is needed; itrequires only a mutantphenotype.

Using cloned DNA

Identification of nucleic acids by hybridization (Southern, Northern)DNA sequence determination

Identification of nucleic acids by hybridization

Mixtures of different-sized DNA or RNA fragments can be separated by gelelectrophoresis.

M 1 2 3 4 5 M

Vector

Identification of nucleic acids by hybridization (DNA = Southern, RNA = Northern)

Southern analysis

left: EtBr-stained gelright: autoradiogram

Plasmids containing different DNAinserts were digested with arestriction enzyme and seperated bygel electrophoresis. Theautoradiogram shows these insertsthat share sequence homology withthe probe.

Comparison of Southern, Northern andWestern analysis of Gene X.

DNA sequencing: the Maxam-Gilbert method

G A/G C/T C

The Sanger Method

uses "stop" nucleotides for sequence

The Sanger Method

DNA-SequenzierungDNA-Sequenzierung nach der Kettenabbruchmethode (Sanger 1977)

Didesoxynucleotide: werden von DNAPolymerasen eingebaut,führen aber anschließendzum Kettenabbruch

H

ddCTP

Primer

viele Moleküle statistischKettenabbrüche an jeder Position

Vier parallele Reaktionsansätze:

GATC

DNA-Sequenzierung:FluoreszenzAutomatisierte nicht-radioaktive DNA-Sequenzierung

Dye Terminator Technologie: jedes der vier Didesoxynucleotide ist chemischmit einem individuellen Fluoreszenzfarbstoff gekoppelt.

für jede Position wird ein Kettenabbruchfragment erzeugt, dessen Fluoreszenz-Lichtfarbe die entsprechende Base anzeigtalle 4 Sequenzierreaktionen werden in EINER Gelspur getrennt

Small DNA molecules can be chemically synthesized(for example PCR primers)

Synthetic DNAis useful for:generatingpolylinkersequences,sequencingDNA, isolatingclones ofinterest,creating site-specificmutations

Oligonucleotides are synthesized using solid-phase synthesis andphosphoramidite nucleoside chemistry. Synthesis proceeds from the3' to the 5' end. The 3' nucleoside is already coupled to the solidsupport (polystyrene or glass), and activated nucleotides aresequentially added on to this nucleoside. Bases and phosphategroups are kept protected until the end of synthesis. At each step,the sugar protective dimethyl trityl (DMT) group on the deoxyriboseis removed to allow reaction with the next amidite.

Analysis of DNA sequences:any piece of DNA has 6 possible reading frames.

Detection of disease-associated alles

Applications

• Early detection of disease-associated alleles– fetal cells obtained by amniocentesis or chorionic villus

sampling– combinations of PCR, restriction digestion, Southern blotting,

and direct sequencing can identify homozygotes as well asheterozygotes

– can identify SNPs, single nucleotide polymorphisms• Genetic engineering

– use of recombinant DNA technology to alter genotype oforganism

– engineered genes called transgenes are used to constructtransgenic organisms

– numerous applications in addition to study of genes

Detection of the sickle-cell globin gene by Southern blotting

Recombinant DNA technology in eukaryotes.

Vectors for yeast

Two ways of integration in a recipient yeast strain

Genetic engineering: fungi

• Mainly the yeast Saccharomyces cerevisiae• Integrative plasmids (YIps)

– derived from bacterial plasmids– have yeast sequences that facilitate homologous

recombination with yeast chromosomes• Autonomously replicating vectors, e.g., shuttle vectors

with both bacterial and yeast origins of replication• Yeast artificial chromosomes (YACs)

– derived from plasmid with centromere and origin ofreplication

– widely used for cloning large genomes

In the process of causing crown gall disease, the bacteriumAgrobacterium tumefaciens inserts a part of its Ti (tumor inducing)plasmid - a region called T-DNA into a chromosome of the host plant.

The Ti plasmid of A. tumefaciens

Ti plasmid

Agrobacteria mediated plant transformation

Genetic engineering: plants

• Considerable agricultural importance• Considerable controversy regarding health and environmental

safety– recombinant plants often referred to as GMOs, genetically

modified organisms– argument that long-term effects are unknown

• Two major methods for transformation– Ti plasmid from Agrobacterium tumefaciens

• upon infection of plant with bacteria containingrecombinant Ti plasmids, plasmids are transferred andthe T-DNA is inserted into host plant genome

• plasmid itself is genetically modified to include polylinker(multiple restriction sites) and drug resistance genes

– gene gun to inject DNA-coated micropellets into cells

T-DNA and any DNA contained within it are inserted into a plantchromosome and are transmitted in a Mendelian pattern of inheritance.

hemizygous state

Creation of C. elegans transgenes

Creation of D. melanogaster transgenes

Creation of M. musculus transgenes

Expressing eukaryotic genes in bacteria

Transgenes and transgenic organisms

pure product

Production of a protein in the milk oftransgenic sheep

Message

Fungal, plant, and animal genes can be clonedand manipulated in bacteria and reintroducedinto eukaryotes, where they generallyintegrate into chromosomes.

Reverse Genetics: gene knockout

Producing a knockout mouse

Transgenic mouse. The two mice are siblings. The left is transgenic for the rat growthhormone gene under the control of the mouse metallothionein promoter. In thepresence of heavy metals, this promotor is induced, resulting in enhanced growth ofthe transgenic mouse.

Genetic engineering: animals

• Numerous model systems and applications• D. melanogaster

– transformation using plasmids derived from Ptransposable elements

• one recombinant containing ends of P elementneeded for insertion flanking the cloned DNA

• one containing P transposase to allow integration– injected into posterior pole of syncitial egg– recombinant vector integrates into host chromosomes– genes recovered in F1 progeny of injected individuals

Genetic engineering: mice

• Technology developed for mice is potentially applicable tohumans

• Ectopic insertions and gene targeting– pros and cons to each method

• e.g., ectopic insertion may place gene inchromosomal location where its expression isaffected, phenomenon called position effect

– targeted gene replacement is common tool• gene knockout (KO) replaces active gene in entire

organism with inactive version• KO often prepared using genetically modified

embryonic stem cells