LECTURE TOPICS:

• RESTRICTION ENZYMES
• GEL ELECTROPHORESIS OF DNA
• DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS
• RECOMBINANT GENE CONSTRUCTION AND CLONING
• CLONING VECTORS, EXPRESSION OF CLONED GENES
• MAKING AND SCREENING GENE LIBRARIES
• POLYMERASE CHAIN REACTION (PCR)
• SITE-SPECIFIC MUTAGENESIS: PROTEIN DESIGN

• Recombinant DNA technology (about 20 years old now) is an unbelievably powerful tool for gene manipulation. The methods associated with this "technology" have made genetic engineering a reality. DNA (genes), RNA, and protein structure and function can be altered by design for beneficial (or detrimental -biological warfare?) results.

• Key methods or tools which are used:
• Enzymes which cut, join and replicate DNA (or RNA-reverse transcriptase)

• Base-pairing - utilization of observation that single-stranded DNA molecules which are complementary will form base-paired structures even if the number of base pairs is small (2 or 3 - like ends of restriction fragments)

• The hybridization (DNA-DNA, DNA-RNA) method is almost always used in one way or another to construct new combinations of DNA fragments and to detect particular DNA or RNA sequences.

• Plasmids (accessory chromosomes) as DNA vectors

• Viruses of bacteria or eucaryotic cells to use as vectors to deliver recombinant DNA

• DNA sequence determination methods (millions of bases of DNA sequences already known)

• DNA synthesis methods to make parts of genes in test tube

• Gene modification procedures.

RESTRICTION ENZYMES (ENDONUCLEASES)

• DNA scissors - hundreds of restriction enzymes are known

• Recognition sequences are of different length (4-8bp), palindromic (have two-fold rotational axis of symmetry), specific cleavage sites [Fig. 6-2]

• They can leave overhanging ends or blunt ends

• Named (ex: HindIII) for the bacterial strain from which it is obtained:

• The number of cuts produced in a specific DNA molecule by a given restriction enzyme 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.

• Agarose or acrylamide gel electrophoresis can resolve DNA restriction fragments which can be visualized by staining or by autoradiography (acrylamide for up to 1,000bp and agarose for up to 20kbp). [Fig. 6-3]

• Even differences of one base pair can be detected on gels. [Illustrations and Fig. 6-6]

• DNA bands on gels can be transferred (Southern blotting) to nitrocellulose filters and identified by hybridization with a specific gene probe (p. 119). [Fig. 6-4]

• Southern (DNA), Northern (RNA), and Western (protein) blotting methods are all powerful probes of gene function.

• Restriction fragment length polymorphism (RFLP) gel analysis is a powerful diagnostic tool (ex.: sickle-cell anemia [Fig 7-52] genetic typing for uniqueness of gene "fingerprint"; O.J Simpson case).

• All methods rely on controlled random reactions which assure an equimolar collection of reaction products. A reaction-specific for each base must occur with equal frequency each time the base occurs in a DNA molecule.

(SANGER DIDEOXY METHOD)         [Fig. 6-7a, Fig. 6-7b]

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

        b) 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).

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

        d) Again, run reaction products on gel, autoradiograph, and read sequence directly. 500-600 bases can be
            easily read on one gel.

If fluorescent-labelled nucleotides are used (a different color for each base), the reaction products can be detected by automated fluorometers [Fig. 6-8] and analyzed by a computer immediately. This is how enormous amounts of data are currently taken for the human genome project. time.

DNA sequencing is also possible by detecting hybridization signals on arrays of short DNA sequences bound to microchips [p.126].

2) LANDMARK SEQUENCES COMPLETED

 

tRNA - (1964) - 75 bases (old, slow, complicated method) First complete DNA genome: X174 DNA (1977) - 5386 bases;. human mitochondrial DNA (1981) - 16,569 bases tobacco chloroplast DNA (1986) - 155,844 bases First complete bacterial genome (H. Influenzae) - 1.9X106 bases E. coli (199?) 3X106 bases [almost done in 1995] human genome (2001?) - 3X109 bases [1995 - 1X106 bases per month!!!!]

DNA SYNTHESIS         [Blocked Nucleotide, Fig. 6-9]

• Chemical synthesis of short oligonucleotides and parts of genes by solid phase, automated methods aid in DNA and RNA sequencing, cloning, site-directed mutagenesis, and gene probing by hybridization. Costs less than $2/base. Start synthesis with blocked nucleotide linked to a solid support. Activated monomers (deoxyribonucleoside 3'-phosphoramidites) which are blocked at 5'-P (and have protected amino groups) are added stepwise to get desired sequence of easily up to 100 nucleotides long (15-30 used most often)

• these chemically synthesized probes are key to facilitate protein engineering

* Novel combinations of genes can be constructed, cloned, amplified and expressed in foreign environments.
 

KEY METHODS OR TOOLS:

• DNA SEQUENCING AND SYNTHESIS METHODS
• ENZYMES to cut, join and replicate DNA
• VECTORS to deliver recombinant DNA
• GEL ELECTROPHORESIS OF DNA
• BASE PAIRING (hybridization) of complementary polynucleotides

• Construct recombinant molecule (chimeric DNA) by covalently linking a chosen DNA insert with a vector which can replicate autonomously in a host cell. [Fig. 6-12]

• Amplify DNA - Introduce DNA into host cells by uptake of naked DNA or DNA incorporated into virus particle.

• Selection by antibiotic resistance, gene probing, antibody reaction

• Restriction enzymes are used to cut vectors at a unique site into which is cloned a DNA insert which has ends like those of the restriction fragment. The vector and insert are covalently linked with DNA ligase. Sometimes synthetic linkers are used to create the necessary restriction sites [Fig. 6-13]

• Plasmids (accessory chromosomes which replicate autonomously) are excellent vectors for cloning 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. [Fig. 6-14]

• Special mutant lambda phages can be used to clone large pieces (10-20kb) of DNA in between the ends of lambda DNA. Especially suitable for cloning of libraries of cDNA or eucaryotic genomic DNA . [Fig. 6-16, Fig. 6-19]

• Can clone large pieces of DNA (100,000 to a million base pairs) in Yeast artificial chromosomes (YACs) [Fig 6-21]

• M13 phage can be used to clone DNA and is especially useful for DNA sequencing of a few thousand base pairs. DNA is cloned into restriction sites in the double-stranded replicative form (RF). [Fig. 6-18]

• LIBRARIES of cloned genes are a collection of cloned sequences which represents a whole genomes.

                        b) cDNA library - made from total cellular mRNA mixtures [ p. 136, Fig. 6-28]. Use reverse
                            transcriptase to get DNA, etc

SCREENING GENE LIBRARIES (Identification of specific sequences (genes) - like looking for a needle in haystack!)
 

• About a half million clones must be screened to find a specific sequence in a genomic library. Easier for abundant mRNA molecules in a cDNA library.

• Hybridization screening - transfer plaques (lambda phage cloning) or bacterial colonies (plasmid cloning) to nitrocellulose filters, denature DNA, hybridize to a DNA or RNA gene probe, autoradiography or filter. Dark spots identify recombinant vectors - MUST have a probe for a gene! [Fig. 6-20]

• Synthetic DNA probes can be made after reverse translation on paper of a protein sequence to predict the DNA sequence coding for a polypeptide. Then hybridize to filter blots of library. [p. 131]

• Immunochemical screening with antibodies is also used [ Fig. 6-29].

• CHROMOSOME WALKING allows long regions of a chromosomal DNA to be explored by successive hybridization, subcloning, and rescreening. DNA fragments near the ends of an original clone are used to identify longer clones which contain their sequence and adjacent sequences extending past theoriginal DNA's ends. [Fig. 6-22]

• An incredibly powerful method for amplifying DNA from very small amounts. Two oligonucleotides (which span the gene of interest and are on opposite strands) are used to prime multiple cycles of DNA sythesis catalyzed by a heat stable DNA polymerase (ex., Taq polymerase from a thermophilic bacterium).[PCRa] Sufficient amounts of DNA are synthesized in 25-40 cycles of replication for cloning or direct DNA sequencing. [PCRb]

• Useful in forensics, diagnosis of genetic defects in utero, sequencing DNA from fossils, etc.

• Expression vectors are designed to give efficient transcription and translation by cloning a gene near a strong bacterial promoter (or eucaryotic promoter, if using a eucaryotic vector). The gene must also be cloned in the correct reading frame with a properly spaced ribosome binding site. A bit of the 5'-end (N-terminus) of an easily screened bacterial gene is often used (Ex: -galactosidase) - screening is for insertional activation(white vs blue colonies or plaques). Immunochemical screening will identify clones, if an antibody to the cloned protein is available.

• vector delivery - genes are introduced by transformation with (1)calcium phosphate precipitated DNA; (2)microinjection; (3) virus vectors(SV40); (4) microprojectiles coated with DNA; (5) liposomes; (6) electroporation, etc. Retroviruses (ex: Maloney murine leukemia virus) are good vectors [Fig. 6-26-31]; the cloned gene (up to 6kb) can be integrated into the host's chromosomes.

• Proinsulin cDNA was cloned in a plasmid and the proinsulin was made by E. coli cells. Many other cDNAs for specific genes have been cloned and expressed this way. This is a basisof the medical biotechnology industry. [Fig. 6-27]

• 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 with genes cloned into the Ti-plasmid of Agrobacterium tumefaciens. Part (the T-DNA) of this plasmid are 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. [Fig. 6-34]

• Strategy is to change the coding information of a gene to get a different amino acid sequence.

• Cut with a restriction enzyme, then chew away one or more bases and religate. [see illustrations]

• Use a synthetic oligonucleotide with a mismatched base, hybridize at low stringency to normal gene (single-stranded), then synthesizing rest of complementary strand, clone and express as new protein [Fig. 6-37].

• Use cassette mutagenesis [Fig.6-38] to introduce a DNA fragment with one or more changes from normal gene.

• Can start with a DNA sequence and ultimately isolate an unknown protein. Also can start with a known protein and isolate its gene. [Fig. 6-39].

• Chromosome mapping and sequencing (even whole human genome).

• Molecular basis of development, evolutionary relationships

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

• human hormone synthesis in bacteria

• antiviral agents (interferon)

• 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 deliver with retroviruses 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

• O.J. Simpson case