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Chapter 5 Notes Biochemistry 461 Fall 2010

CHAPTER 5, EXPLORING GENES:

LECTURE TOPICS

1) RESTRICTION ENZYMES

2) GEL ELECTROPHORESIS OF DNA

3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS

4) POLYMERASE CHAIN REACTION (PCR)

5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING

6) DNA CLONING VECTORS

7) GENE LIBRARIES: MAKING AND SCREENING THEM

8) CHROMOSOME MAPPING

9) EXPRESSION OF CLONED GENES

10) ENGINEERING NOVEL PROTEINS

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Recombinant DNA technology (started in late 1970's)

! An incredibly powerful set of tool for gene manipulation.

! Methods associated with this "technology" make genetic engineering areality.

! DNA (genes), RNA, and protein structure and function can be altered bydesign for beneficial (or detrimental -biological warfare/terrorism?) results.

KEY TOOLS and METHODS OF GENE EXPLORATION

! ENZYMES to cut, join and replicate DNA in test tubes (in vitro)a) restriction enzymes are DNA cuttersb) DNA ligases are DNA joinersc) DNA replication requires DNA polymerases

! GEL ELECTROPHORESIS to separate and isolate specific DNAs

! BLOTTING METHODS based on hybridization (BASE-PAIRING) ofcomplementary DNA and/or RNA

! SOLID PHASE methods to sequence and synthesize DNA

! POLYMERASE CHAIN REACTION for gene detection and amplification.

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RESTRICTION ENZYMES (ENDONUCLEASES)

! DNA scissors - hundreds of restriction enzymes are known

! Recognition sequences - different lengths (often 4-8bp), palindromic (2 -foldrotational axis of symmetry), specific cleavage sites (Fig. 6.1)

! They can leave overhanging ends or blunt ends

! Named (ex: HindIII) for source bacterialstrain:

H = Haemophilus in = influenzaed = strain d III = third one identified

! Number of cuts in a specific DNA ranges fromfew (if long recognition site) to many (short or ambiguous recognition site)

! patterns of fragments are diagnostic of a given DNA species and physical mapsof whole chromosomes can be made. (Fig.6.-2)

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GEL ELECTROPHORESIS OF DNA

! Agarose gels separate DNA restriction fragments

! Visualize DNA by staining or autoradiography

! even differences of one base pair can be detected on gels.

! Hybridization - Base-pairing of DNA-DNA, DNA-RNA: Complementary single-stranded DNA and RNA molecules form base-paired structures even if only 2 or3 bases can pair - like at ends of restriction fragments!

[IMPORTANT: Hybridization (DNA-DNA, DNA-RNA) is almost always used inone or more ways to to detect particular DNA or RNA sequences and toconstruct new combinations of DNA fragments.]

NUCLEIC ACID BLOTTING AND HYBRIDIZATION: DNA bands (and patterns) on gels

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can be transferred to nitrocellulose filters (Southern blotting) and identified byhybridization with a specific gene probe. (Fig.6.3)

! Southern (DNA), Northern (RNA), and Western (protein) blotting methods areall powerful probes of gene function.

! Rest rictionfragment length polymorphism (RFLP) gel analysis is a powerful diagnostictool (ex. sickle-cell anemia genetic typing)

MstI RFLP for Sickle-Cell Mutation Detection [Fig 7-52]

Normal Sickle cell

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DNA SEQUENCING: ALL METHODS REQUIRE THE FOLLOWING*

! reactions specific for each base

! controlled random reactions

! equimolar collection of reaction products (same frequency ofstopping each time the same base occurs)

LANDMARK DNA SEQUENCES COMPLETED

tRNA - (1964) ( , complicated method)5386 bases �X174 DNA (1977) 155,844 bases tobacco chloroplast DNA (1986)1.8 million bases H. influenzae (1995)3 million bases E. coli (1997)

bases human (2000!!)

DNA SEQUENCING BY CONTROLLED RANDOM CHAIN TERMINATION OF DNASYNTHESIS METHOD (SANGER DIDEOXY METHOD) (Fig.6.4)

! Use a template-primer complex, a DNA polymerase, dNTPs, and one 2'-3'dideoxynucleoside triphosphate (one for each base) in each of four reactions.

! DNA synthesis occurs (specific for each base) until a dideoxy nucleosidephosphate is inserted into the nascent DNA - then the reaction stops! (no free3'-OH group to attack the incoming dNTP substrate).

! Reaction is carried out under conditions (adjust ratio of dNTP: ddNTP) to giveequal representation and distribution of products.

! Run reaction products on gel. 500-600 bases can be easily read on one gel.

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Strategy for Chain termination DNA sequencing: (Fig.6.4)

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Current methods for DNA/RNA sequence determination:

! Use fluorescent-labelled nucleotides (makes each base reaction mix flourescea different color); mix all 4 reaction products, and detect all with automatedfluorometers to detect DNA reaction products and analyze by computerimmediately.(Fig. 6.5) [Can sequence whole genomes (ex. Fig.6.6 - bacterial)

! Can also sequence DNA by detecting hybridization signals on arrays of shortDNA sequences bound to microchips.

! RNA SEQUENCING - RNA can be sequenced directly, but now it is done bydideoxy method, using reverse transcriptase with a synthetic DNA primer.

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CHEMICAL SYNTHESIS OF DNA (SOLID PHASE, AUTOMATED METHODS)

! oligodeoxynucleotide chain (short DNA, parts of genes for probes and primers)

! aid DNA/RNA sequencing, cloning, and gene probing by hybridization

! easy to make DNA 100 nucleotides long (18-20 used most often)

! Chemically synthesized DNAs are key to protein engineering by site-directedmutagenesis.

! Start with blocked nucleotide linkedto a solid support (glass bead).

! protected nucleotides addedstepwise (but 3' to 5' - reverse ofDNA polymerase)

! Stepwise linking of activatedmonomers (deoxyribonucleoside 3'-phosphoramidites) that are blockedat 5'-P (and have protected aminogroups)

Chemical synthesis of DNA: (Fig.6.7)

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**See Fig 6.9 fordrawing of three

cycles of PCR

POLYMERASE CHAIN REACTION (PCR) Discovered in 1984

! [K.B. Mullis,Scientific American,1990, 262:56-65)]

! Incredibly powerful method to amplify (synthesize) DNA starting with as little asone copy of a DNA molecule. (Figs.6.8 and 6.9)

! PCR CONCEPT (Fig.6.8): Two oligonucleotidesspanning a gene of interest and on oppositestrands are used to prime multiple cycles of DNAsynthesis catalyzed by a heat stable DNApolymerase (ex., Taq polymerase from athermophilic bacterium).

Ex:PCR starting with one of 2 strands of DNA.

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! PCR RESULT: In 25-40 replication cycles, enough DNA is synthesized toclone or to sequencedirectly.

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! PCR USES: Forensics, diagnosis of genetic defects in utero, sequencing DNAfrom fossils, detecting small amounts of bacteria (ex; anthrax), etc.

! FORENSICS EXAMPLE: Bloodstain analysis of victim (V) and clothing ofdefendant (D - apparently guilty!). (Fig.6.10)

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CONSTRUCTION, CLONING AND EXPRESSION OF DNA: Novel combinations of genes can be constructed, cloned, amplified andexpressed in foreign environments.

KEY STEPS/PROCEDURES/TOOLS [Fig.6.11, 4th Ed.]

! Construct recombinant molecule (link DNA insert with a vector).

! Amplify and clone DNA - Introduce DNA into host cells as naked DNA or asDNA incorporated into virus particle. DNA must be replicated autonomously in ahost cell.

! Selection by antibiotic resistance, gene probing, antibody reaction

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CONSTRUCT RECOMBINANT MOLECULE - link a DNA insert with a vector.(Figs.6.11,12)

CUT AND JOIN DIFFERENT DNA MOLECULES: Restriction enzymes and DNAligase.

! Restriction enzymes that give “cohesive ends” (short complementary endsthat can base pair) cut unique cloning sites in vectors.[Ex: Eco RI (Fig.6.11)]

The vector and insert are covalently linked with DNA ligase.

! Restriction enzymes that give “blunt ends” need “linkers” added by T4DNA ligase to get “cohesive ends” for cloning. [Ex: Eco RI linker (Fig.6.12)]

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Amplify and clone DNA: Introduce DNA into host cells as naked DNA [Seeillustration page 13] or as DNA incorporated into virus particle. DNA must bereplicated autonomously in a host cell.

Selection of recombinant DNA-containing host cells - antibiotic resistance, geneprobing, antibody reactions, etc.

SOME CLONING VECTORS (autonomously replicating)

Plasmids: Insertional inactivation of antibiotic resistance often used. Use forsmall DNAs.

Lambda phages (Larger DNA pieces tha plasmids. Especially useful forlibraries of cDNA or eucaryotic genomic DNA)

YACs and BACs (yeast and bacterial artificial chromosomes) - for long DNA,especially big pieces of chromosomes.

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CLONING VECTORS

! Plasmids (accessory chromosomes whichreplicate autonomously) are excellent vectors forcloning DNA of 2-6 kb in E. coli. Antibioticresistances are used for selection ofrecombinant plasmids. Insertional inactivationsignals the presence of a DNA insert in anantibiotic resistance gene. (See page 15 andFig.6.13)

! Special lambda (�) phages (they work a lot likeT2 bateriophage) used to clone large pieces (10-20kb) of DNA between eachend of lambda DNA. Especially suitable for cloning of libraries of cDNA oreucaryotic genomic DNA. (Fig.6.14,15)

lambda (�) phagelifecycle

Cloning DNAbetween each end oflambda (�) DNA

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! Yeast artificial chromosomes (YACs) - Can clone large pieces of DNA(100,000 to a million base pairs). (Fig.6.21)

GENE LIBRARIES: What are they??

Genomic library. Made from all restriction fragments of a cell's DNA. A collection ofcloned sequences which represents a whole genome.

! Genomic library vectors: Bacteriophage lambda, YACs and BACs

cDNA library. Made from mix of all of a cell’s mRNAs. Use reverse transcriptase to getDNA. A cDNA library is a mix of DNAs complementary to ALL genes thatare being expressed as mRNA’s.

! CDNA library vectors: lambda phage, plasmids

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Constructing a genomic library in bacteriophage �. Made from all restrictionfragments of a cell's DNA. A collection of cloned DNA sequences which representsa whole genome.

Constructing a cDNA library from a cell’s mRNAs. Use reverse transcriptase to getDNA. A cDNA library is a mix of DNAs that represents all of the cell’s mRNAs.

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SCREENING GENE LIBRARIES: searching for a needle in phagestack!

! screen 500,000 clones for a specific sequence in a genomic library! Easier for abundant mRNA molecules in a cDNA library.! Hybridization screening with gene probe! Synthetic DNA probes (predict sequence by reverse translation of protein

sequence to a DNA sequence)! Immunochemical (antibody) screening of an “expression library”! Chromosome walking to connect long pieces fo chromosomes! Can map whole chromosomes - use lambda or YACs for cloning

Screening for a specific gene with a radioactive gene probe. (Or antibodiesor fluorescence)

Screening a cDNA library.

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REVERSE TRANSLATION: Make a gene probe from a known protein sequence.

! Predict DNA base sequence from amino acid sequence, using Genetic Code! Synthesize DNA probes from the predicted gene sequences! This example (Fig.6.20) requires a mix of 256 different oligonucleotides to

guarantee a perfect match. (These mixtures really work!!)

CHROMOSOME WALKING: Use to map/explore long regions of chromosomes byiterative hybridization, subcloning, and rescreening.

! DNA fragments near ends of one clone are used to identify longer clones whichcontain their sequence and adjacent sequences extending past the originalDNAs ends. (Fig.6.22)

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EXPRESSION OF CLONED GENES

DNA vector delivery to cells.

! calcium phosphate precipitated DNA! microinjection! virus vectors(SV40), vaccinia, retroviruses (ex: Maloney

murine leukemia virus)! GENE GUN" (microprojectiles coated with DNA)! liposomes! electroporation

Expression vectors:

! Designed to give efficient transcription and translation by cloning a genenear a strong promoter.

! The gene must be cloned in the correct reading frame with a properlyspaced ribosome binding site.

! Immunochemical screening will identify clones, if an antibody to thecloned protein is available.

SOME EXAMPLES

! Proinsulin cDNA was cloned in a plasmid and the proinsulin was made by E.coli cells. This is a standard method to express cloded genes as proteins, and isthe basis for much of the biotechnology industry. (Fig.6.23)

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! Genetically engineered giant mice result from injection of somatotropin geneinto male pronucleus of a fertilized mouse egg. Cd++ controls expression of thisgene by its placement under control of the metallothionein gene. [Fig. 6-32]

! Plant Genetic Engineering. Genes are cloned into the Ti-plasmid ofAgrobacterium tumefaciens. Part of this plasmid (the T-DNA) is incorporatedinto plant chromosomes after Agrobacterium infection. DNA which is clonedbetween the right and left ends of the T-DNA can be expressed by "transformed"plant cells after integration in a chromosome. (Figs.6.33,34)

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Ti-plasmid of Agrobacterium tumefaciens

Gene disruption/replacement. Genes can be inactivated (“knockout” mutant) orreplaced by a modified or completely new gene by homologous recombination

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ENGINEERING NOVEL PROTEINS:

! Modify DNA coding information to get protein with different amino acid sequence.

! These are really novel combinations which would not occur in nature.

! Solid phase synthesis of whole genes of any type is now possible.

Site-specific mutagenesis - Hybridize synthetic oligonucleotide with a mismatchedbase (Fig.6.36)

Cassette Mutagenesis: (Fig.6.37)

! Use restriction fragments tocombine parts of genes coding fordifferent domains of differentproteins.

! introduce a DNA fragment withone or more changes from normalgene.

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SUMMARY: Recombinant DNA technology Roadmap

! Can start with a DNA sequence and ultimately isolate an unknown protein. Alsocan start with a known protein and isolate its gene. (Fig. 6-38)

CURRENT AND FUTURE APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY

! Chromosome mapping and sequencing

! Discovery of molecular bases of development, evolutionary relationships

! New proteins with new functions (or old proteins with new functions!)

! human hormone synthesis in bacteria

! antiviral agents

! AIDS vaccine development

! new pharmacological agents (proteins, RNA, DNA)

! antisense RNA therapy

! medical diagnostic reagents (gene probes) for detection of genetic diseases,infections and cancers

! gene delivery with disarmed viruses to alleviate diseases caused by known genedefects.

! agricultural revolution with animals having altered traits, more nutritious plants,heat/drought resistant crops, etc.

! forensics - molecular detectives