LECTURE TOPICS 3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS 5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING

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1 Page 1 of 25 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

2 Page 2 of 25 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 a reality.! DNA (genes), RNA, and protein structure and function can be altered by design 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 cutters b) DNA ligases are DNA joiners c) DNA replication requires DNA polymerases! GEL ELECTROPHORESIS to separate and isolate specific DNAs! BLOTTING METHODS based on hybridization (BASE-PAIRING) of complementary DNA and/or RNA! SOLID PHASE methods to sequence and synthesize DNA! POLYMERASE CHAIN REACTION for gene detection and amplification.

3 Page 3 of 25 RESTRICTION ENZYMES (ENDONUCLEASES)! DNA scissors - hundreds of restriction enzymes are known! Recognition sequences - different lengths (often 4-8bp), palindromic (2 -fold rotational axis of symmetry), specific cleavage sites (Fig. 6.1)! They can leave overhanging ends or blunt ends! Named (ex: HindIII) for source bacterial strain: H = Haemophilus in = influenzae d = strain d III = third one identified! Number of cuts in a specific DNA ranges from few (if long recognition site) to many (short or ambiguous recognition site)! patterns of fragments are diagnostic of a given DNA species and physical maps of whole chromosomes can be made. (Fig.6.-2)

4 Page 4 of 25 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 singlestranded DNA and RNA molecules form base-paired structures even if only 2 or 3 bases can pair - like at ends of restriction fragments! [IMPORTANT: Hybridization (DNA-DNA, DNA-RNA) is almost always used in one or more ways to to detect particular DNA or RNA sequences and to construct new combinations of DNA fragments.] NUCLEIC ACID BLOTTING AND HYBRIDIZATION: DNA bands (and patterns) on gels

5 can be transferred to nitrocellulose filters (Southern blotting) and identified by hybridization with a specific gene probe. (Fig.6.3) Page 5 of 25! Southern (DNA), Northern (RNA), and Western (protein) blotting methods are all powerful probes of gene function.! Rest riction fragment length polymorphism (RFLP) gel analysis is a powerful diagnostic tool (ex. sickle-cell anemia genetic typing) MstI RFLP for Sickle-Cell Mutation Detection [Fig 7-52] Normal Sickle cell

6 Page 6 of 25 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: ALL METHODS REQUIRE THE FOLLOWING*! reactions specific for each base! controlled random reactions! equimolar collection of reaction products (same frequency of stopping each time the same base occurs) DNA SEQUENCING BY CONTROLLED RANDOM CHAIN TERMINATION OF DNA SYNTHESIS 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 nucleoside phosphate is inserted into the nascent DNA - then the reaction stops! (no free 3'-OH group to attack the incoming dntp substrate).! Reaction is carried out under conditions (adjust ratio of dntp: ddntp) to give equal representation and distribution of products.! Run reaction products on gel bases can be easily read on one gel.

7 Strategy for Chain termination DNA sequencing: (Fig.6.4) Page 7 of 25

8 Page 8 of 25 Current methods for DNA/RNA sequence determination:! Use fluorescent-labelled nucleotides (makes each base reaction mix flouresce a different color); mix all 4 reaction products, and detect all with automated fluorometers to detect DNA reaction products and analyze by computer immediately.(fig. 6.5) [Can sequence whole genomes (ex. Fig bacterial)! Can also sequence DNA by detecting hybridization signals on arrays of short DNA sequences bound to microchips.! RNA SEQUENCING - RNA can be sequenced directly, but now it is done by dideoxy method, using reverse transcriptase with a synthetic DNA primer.

9 CHEMICAL SYNTHESIS OF DNA (SOLID PHASE, AUTOMATED METHODS) Page 9 of 25! 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-directed mutagenesis.! Start with blocked nucleotide linked to a solid support (glass bead).! protected nucleotides added stepwise (but 3' to 5' - reverse of DNA polymerase)! Stepwise linking of activated monomers (deoxyribonucleoside 3'- phosphoramidites) that are blocked at 5'-P (and have protected amino groups) Chemical synthesis of DNA: (Fig.6.7)

10 Page 10 of 25 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 as one copy of a DNA molecule. (Figs.6.8 and 6.9)! PCR CONCEPT (Fig.6.8): Two oligonucleotides spanning a gene of interest and on opposite strands are used to prime multiple cycles of DNA synthesis catalyzed by a heat stable DNA polymerase (ex., Taq polymerase from a thermophilic bacterium). Ex:PCR starting with one of 2 strands of DNA. **See Fig 6.9 for drawing of three cycles of PCR

11 Page 11 of 25! PCR RESULT: In replication cycles, enough DNA is synthesized to clone or to sequencedirectly.

12 Page 12 of 25! PCR USES: Forensics, diagnosis of genetic defects in utero, sequencing DNA from fossils, detecting small amounts of bacteria (ex; anthrax), etc.! FORENSICS EXAMPLE: Bloodstain analysis of victim (V) and clothing of defendant (D - apparently guilty!). (Fig.6.10)

13 Page 13 of 25 CONSTRUCTION, CLONING AND EXPRESSION OF DNA: Novel combinations of genes can be constructed, cloned, amplified and expressed in foreign environments. KEY STEPS/PROCEDURES/TOOLS [Fig.6.11, 4 th Ed.]! Construct recombinant molecule (link DNA insert with a vector).! Amplify and clone DNA - Introduce DNA into host cells as naked DNA or as DNA incorporated into virus particle. DNA must be replicated autonomously in a host cell.! Selection by antibiotic resistance, gene probing, antibody reaction

14 CONSTRUCT RECOMBINANT MOLECULE - link a DNA insert with a vector. (Figs.6.11,12) Page 14 of 25 CUT AND JOIN DIFFERENT DNA MOLECULES: Restriction enzymes and DNA ligase.! Restriction enzymes that give cohesive ends (short complementary ends that 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 T4 DNA ligase to get cohesive ends for cloning. [Ex: Eco RI linker (Fig.6.12)]

15 Page 15 of 25 Amplify and clone DNA: Introduce DNA into host cells as naked DNA [See illustration page 13] or as DNA incorporated into virus particle. DNA must be replicated autonomously in a host cell. Selection of recombinant DNA-containing host cells - antibiotic resistance, gene probing, antibody reactions, etc. SOME CLONING VECTORS (autonomously replicating) Plasmids: Insertional inactivation of antibiotic resistance often used. Use for small DNAs. Lambda phages (Larger DNA pieces tha plasmids. Especially useful for libraries of cdna or eucaryotic genomic DNA) YACs and BACs (yeast and bacterial artificial chromosomes) - for long DNA, especially big pieces of chromosomes.

16 Page 16 of 25 CLONING VECTORS! Plasmids (accessory chromosomes which replicate autonomously) are excellent vectors for cloning DNA of 2-6 kb in E. coli. Antibiotic resistances are used for selection of recombinant plasmids. Insertional inactivation signals the presence of a DNA insert in an antibiotic resistance gene. (See page 15 and Fig.6.13)! Special lambda ( ) phages (they work a lot like T2 bateriophage) used to clone large pieces (10-20kb) of DNA between each end of lambda DNA. Especially suitable for cloning of libraries of cdna or eucaryotic genomic DNA. (Fig.6.14,15) lambda ( ) phage lifecycle Cloning DNA between each end of lambda ( ) DNA

17 ! Yeast artificial chromosomes (YACs) - Can clone large pieces of DNA (100,000 to a million base pairs). (Fig.6.21) Page 17 of 25 GENE LIBRARIES: What are they?? Genomic library. Made from all restriction fragments of a cell's DNA. A collection of cloned 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 get DNA. A cdna library is a mix of DNAs complementary to ALL genes that are being expressed as mrna s.! CDNA library vectors: lambda phage, plasmids

18 Page 18 of 25 Constructing a genomic library in bacteriophage. Made from all restriction fragments of a cell's DNA. A collection of cloned DNA sequences which represents a whole genome. Constructing a cdna library from a cell s mrnas. Use reverse transcriptase to get DNA. A cdna library is a mix of DNAs that represents all of the cell s mrnas.

19 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 Page 19 of 25 Screening for a specific gene with a radioactive gene probe. (Or antibodies or fluorescence) Screening a cdna library.

20 Page 20 of 25 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 by iterative hybridization, subcloning, and rescreening.! DNA fragments near ends of one clone are used to identify longer clones which contain their sequence and adjacent sequences extending past the original DNAs ends. (Fig.6.22)

21 Page 21 of 25 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 gene near a strong promoter.! The gene must be cloned in the correct reading frame with a properly spaced ribosome binding site.! Immunochemical screening will identify clones, if an antibody to the cloned 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 is the basis for much of the biotechnology industry. (Fig.6.23)

22 Page 22 of 25! Genetically engineered giant mice result from injection of somatotropin gene into male pronucleus of a fertilized mouse egg. Cd ++ controls expression of this gene by its placement under control of the metallothionein gene. [Fig. 6-32]! Plant Genetic Engineering. Genes are cloned into the Ti-plasmid of Agrobacterium tumefaciens. Part of this plasmid (the T-DNA) is incorporated into plant chromosomes after Agrobacterium infection. DNA which is cloned between 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)

23 Page 23 of 25 Ti-plasmid of Agrobacterium tumefaciens Gene disruption/replacement. Genes can be inactivated ( knockout mutant) or replaced by a modified or completely new gene by homologous recombination

24 Page 24 of 25 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 mismatched base (Fig.6.36) Cassette Mutagenesis: (Fig.6.37)! Use restriction fragments to combine parts of genes coding for different domains of different proteins.! introduce a DNA fragment with one or more changes from normal gene.

25 Page 25 of 25 SUMMARY: Recombinant DNA technology Roadmap! Can start with a DNA sequence and ultimately isolate an unknown protein. Also can 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 gene defects.! agricultural revolution with animals having altered traits, more nutritious plants, heat/drought resistant crops, etc.! forensics - molecular detectives