Section J Section J Analysis and application of cloning Analysis and application of cloning

DNADNA

Section J -Analysis and application Section J -Analysis and application of cloning DNAof cloning DNA

J1 CHARACTERIZATION OF CLONESJ1 CHARACTERIZATION OF CLONES J2 NUCLEIC AND SEQUENCINGJ2 NUCLEIC AND SEQUENCING J3 POLYMERASE CHAIN REACTION J3 POLYMERASE CHAIN REACTION J4 ORGANIZATION OF CLONED J4 ORGANIZATION OF CLONED

GENESGENES J5 MUTAGENESIS OF CLONED GENE J5 MUTAGENESIS OF CLONED GENE J6 APPLICATIONS OF CLONINGJ6 APPLICATIONS OF CLONING

J1 CHARACTERIZATION OF J1 CHARACTERIZATION OF CLONESCLONES

CharacterizationCharacterization Determining Determining various properties of recombina various properties of recombinant DNA molecule, such as size, restriction map, orientation of annt DNA molecule, such as size, restriction map, orientation of any gene present and nucleotide sequence, constitutes the process y gene present and nucleotide sequence, constitutes the process of clone characterization. It requires a purified preparation of the of clone characterization. It requires a purified preparation of the cloned DNA.cloned DNA.

Restriction mappingRestriction mapping Digesting recombinant DNA molecules Digesting recombinant DNA molecules with restriction enzymes, alone and in combination, allows the cwith restriction enzymes, alone and in combination, allows the construction of a diagram(restriction map) of the molecules indiconstruction of a diagram(restriction map) of the molecules indicating the cleavage position and fragment sizes.ating the cleavage position and fragment sizes.

Partial digestionPartial digestion The partial digestion of end-labeled DNA fra The partial digestion of end-labeled DNA fragments with restriction enzymes, and sizing of fragmentsgments with restriction enzymes, and sizing of fragments produc produced, also enables a restriction map to be constructed.ed, also enables a restriction map to be constructed.

Labelilg nucleic acid DNA and RNALabelilg nucleic acid DNA and RNA can be end-labe can be end-labeled using polynucleotide kinase or terminal transferasled using polynucleotide kinase or terminal transferase. Uniform labeling requires polymerases to synthesize. Uniform labeling requires polymerases to synthesize a complete labeled strand.e a complete labeled strand.

Southern and Northern blottingSouthern and Northern blotting The nucleic acid i The nucleic acid in lanes of a gel is transferred to a memebrane, bound n lanes of a gel is transferred to a memebrane, bound and then hybridized with a labeled nucleic acid probe. and then hybridized with a labeled nucleic acid probe. Washing removes nonhybridized probe, and the memWashing removes nonhybridized probe, and the membrane is then treated to reveal the bands produced. Spbrane is then treated to reveal the bands produced. Specific RNA species are detected on Northern blots, wecific RNA species are detected on Northern blots, whereas the DNA bands on Southern blots could be gehereas the DNA bands on Southern blots could be genes in genomic DNA or parts of cloned genes.nes in genomic DNA or parts of cloned genes.

Fig. 1. Restriction mapping (a) using single and double digests to Fig. 1. Restriction mapping (a) using single and double digests to completion; (b) by partial digestion of an end-labeled molecule. * is completion; (b) by partial digestion of an end-labeled molecule. * is

the labeled endthe labeled end

Fig. 2. 5′-End labeling of a Fig. 2. 5′-End labeling of a nucleic acid molecule.nucleic acid molecule.

Fig. 3. Southern Fig. 3. Southern blotting.blotting.

J2 NUCLEIC AND J2 NUCLEIC AND SEQUENCINGSEQUENCING

DNA sequencingDNA sequencing The two main methods of DNA seqThe two main methods of DNA sequencing are Maxam and Gilbert chemical method in whuencing are Maxam and Gilbert chemical method in which end-labeled DNA is subjected to base-specific cleavich end-labeled DNA is subjected to base-specific cleave reaction prior to gel separation, and Snger’s enzymic e reaction prior to gel separation, and Snger’s enzymic method. The latter uses dideoxynucleotides as chain termethod. The latter uses dideoxynucleotides as chain terminators to produce a ladder of molecules generated by minators to produce a ladder of molecules generated by polymerase extension of primer.polymerase extension of primer.

RNA sequencingRNA sequencing A set of four Rnase that cleave 3’ to A set of four Rnase that cleave 3’ to specific nucleotides are used to produce a ladder of fragspecific nucleotides are used to produce a ladder of fragments from end-labeled RNA. Polyacrylamide gel electments from end-labeled RNA. Polyacrylamide gel electrophoresis analysis allows the sequence to be readrophoresis analysis allows the sequence to be read..

Sequence databasesSequence databases Newly determined DNA ,RNA and Newly determined DNA ,RNA and protein sequences are entered into databases(EMBL and protein sequences are entered into databases(EMBL and GeneBank). These collections of all known sequences are GeneBank). These collections of all known sequences are available for analysis by computer.available for analysis by computer.

Analysis of sequencesAnalysis of sequences Special computer software is used Special computer software is used to search nucleic acid and protein sequences for the preseto search nucleic acid and protein sequences for the presence of or similaritiesnce of or similarities

Genome sequencing projectsGenome sequencing projects The entire genome sequenThe entire genome sequences of several organisms have been determined and those ces of several organisms have been determined and those of other organisms are in progress. Often a genetic map is of other organisms are in progress. Often a genetic map is first produced to aid the project.first produced to aid the project.

Fig. 1. DNA sequencing. (a) An example of a Maxam and Gilbert sequencFig. 1. DNA sequencing. (a) An example of a Maxam and Gilbert sequencing gel; (b) Sanger sequencing.ing gel; (b) Sanger sequencing.

J3 POLYMERASE CHAIN REACTIONJ3 POLYMERASE CHAIN REACTION

The PCR cycleThe PCR cycle The reaction cycle comprises a 95 step to ℃ The reaction cycle comprises a 95 step to ℃denature the duplex DNA, an annealing step of around 55 t℃denature the duplex DNA, an annealing step of around 55 t℃o allow the primers to bind and a 72 polymerization step. ℃o allow the primers to bind and a 72 polymerization step. ℃Mg2+ and dNTP are required in addition to template, primers,Mg2+ and dNTP are required in addition to template, primers, buffer and enzyme. buffer and enzyme.

TemplateTemplate Almost any source that contains one or more intac Almost any source that contains one or more intact target DNA molecule can, in theory, be amplified by PCR, pt target DNA molecule can, in theory, be amplified by PCR, providing appropriate primers can be designed.roviding appropriate primers can be designed.

PrimersPrimers A pair of oligonucleotides of about 18-30 nt with si A pair of oligonucleotides of about 18-30 nt with similar G+C content will serve as PCR primers as long as they milar G+C content will serve as PCR primers as long as they direct DNA synthesis towards one another. Primers with somdirect DNA synthesis towards one another. Primers with some degeneracy can also be used if the target DNA sequence is e degeneracy can also be used if the target DNA sequence is not completely known.not completely known.

EnzymesEnzymes Thermostable DNA polymerases(e.g. Taq pol Thermostable DNA polymerases(e.g. Taq polymerase) are used in PCR as they survive the hot denaturymerase) are used in PCR as they survive the hot denaturation step. Some are more error-prone than others.ation step. Some are more error-prone than others.

PCR optimizationPCR optimization It may be necessary to vary the anne It may be necessary to vary the annealing temperature and/or the Mg2+ concentration to obtaling temperature and/or the Mg2+ concentration to obtain faithful amplification. From complex mixtures, a secain faithful amplification. From complex mixtures, a second pair of nested primers can improve specificity.ond pair of nested primers can improve specificity.

PCR variationPCR variation Variation on basic PCR include quantita Variation on basic PCR include quantitative PCR, degenerate oligonucleotide primer PCR(DOP-tive PCR, degenerate oligonucleotide primer PCR(DOP-PCR), inverse PCR, multiplex PCR, rapid amplification PCR), inverse PCR, multiplex PCR, rapid amplification of cDNA ends(RACE) and PCR mutagenesis.of cDNA ends(RACE) and PCR mutagenesis.

Fig. 1. The first three cycles of a polymerase chain reaction. Only after cycle 3 Fig. 1. The first three cycles of a polymerase chain reaction. Only after cycle 3 are there any duplex molecules which are the exact length of the region to be are there any duplex molecules which are the exact length of the region to be amplified (molecules 2 and 7). After a few more cycles these become the major amplified (molecules 2 and 7). After a few more cycles these become the major

product.product.

J4 ORGANIZATION OF CLONED J4 ORGANIZATION OF CLONED GENESGENES

OrganizationOrganization The polarity of oligo(dT)-primed cDNA clones is often The polarity of oligo(dT)-primed cDNA clones is often apparent from the location of the poly(A), and the coding region can thapparent from the location of the poly(A), and the coding region can thus be deduced. The presence and polarity of any gene in a genomic clous be deduced. The presence and polarity of any gene in a genomic clone is not obvious, but can be determined by mapping and probing expene is not obvious, but can be determined by mapping and probing experiments.riments.

Mapping cDNA on genomic DNAMapping cDNA on genomic DNA Southern blotting, using probes fr Southern blotting, using probes from part of a cDNA clone, can show which parts of a genomic clone haom part of a cDNA clone, can show which parts of a genomic clone have corresponding sequences.ve corresponding sequences.

S1 nuclease mappingS1 nuclease mapping The 5’- or 3’- of a transcript can be identified b The 5’- or 3’- of a transcript can be identified by hybridizing a longer, end-labeled antisense fragment to the RNA. Thy hybridizing a longer, end-labeled antisense fragment to the RNA. The hybrid is treated with nuclease S1 to remove single-stranded regions, e hybrid is treated with nuclease S1 to remove single-stranded regions, and remaining fragment’s size is measured on a gel.and remaining fragment’s size is measured on a gel.

Primer extensionPrimer extension A primer is extended by a polymerase until the end A primer is extended by a polymerase until the end of the template is reached and the polymerase dissociates. The length oof the template is reached and the polymerase dissociates. The length of the extended product indicates the 5’-end of the template.f the extended product indicates the 5’-end of the template.

Gel retardationGel retardation Mixing a protein extract with a labeled D Mixing a protein extract with a labeled DNA fragment and running the mixture on a native gel will sNA fragment and running the mixture on a native gel will show the presence of DNA-protein complexes as retarded bhow the presence of DNA-protein complexes as retarded bands on the gel.ands on the gel.

Dnase I footprintingDnase I footprinting The ‘footprint’ of a protein bound sp The ‘footprint’ of a protein bound specifically to a DNA sequence can be visualized by treating ecifically to a DNA sequence can be visualized by treating the mixture of end-labeled DNA plus protein with small amthe mixture of end-labeled DNA plus protein with small amounts of Dnase I prior to running the mixture on a gel. The ounts of Dnase I prior to running the mixture on a gel. The footprint is a region with few bands in a ladder of cleave prfootprint is a region with few bands in a ladder of cleave products.oducts.

Reporter genesReporter genes To verify the function of promoter, it can To verify the function of promoter, it can be joined to the coding region of an easily detected gene(rebe joined to the coding region of an easily detected gene(reporter gene) and the protein product assayed under conditioporter gene) and the protein product assayed under condition when the promoter should be active.n when the promoter should be active.

Fig. 1. S1 nuclease mapping the 5′-end of an RNA. * = Fig. 1. S1 nuclease mapping the 5′-end of an RNA. * = position of end label.position of end label.

3’DNA 3’

RNA 5’DNA 3’

Add S1 nuclease

* 5’

3’5’

PAGE Analysis

RNA 5’

Fig. 2. Primer extension. * = Fig. 2. Primer extension. * = position of end label.position of end label.

J5 MUTAGENESIS OF CLONED J5 MUTAGENESIS OF CLONED GENEGENE

Deletion mutagenesisDeletion mutagenesis Progressively deleting DNA from one end is very Progressively deleting DNA from one end is very useful for defining the important of particular sequences. Unidirectional duseful for defining the important of particular sequences. Unidirectional deletion can be created using exonuclease III which removes one ztrand in eletion can be created using exonuclease III which removes one ztrand in a 3’ to 5’ direction from a recessed 3’-end. A single strand –specific nuclea 3’ to 5’ direction from a recessed 3’-end. A single strand –specific nuclease then creates blunt end molecules for ligation, and transformation generase then creates blunt end molecules for ligation, and transformation generates then deleted clones.ates then deleted clones.

Site-directed mutagenesisSite-directed mutagenesis Changing one or a few nucleotides at a partic Changing one or a few nucleotides at a particular site usually involves annealing a mutagenic primer to a template folloular site usually involves annealing a mutagenic primer to a template followed by complementary strand synthesis by a DNA polymerase. Formerly. wed by complementary strand synthesis by a DNA polymerase. Formerly. Single-stranded templates prepared using M13 were used, but polymerase Single-stranded templates prepared using M13 were used, but polymerase chain reaction(PCR) techniques are now preferred.chain reaction(PCR) techniques are now preferred.

PCR mutagenesisPCR mutagenesis By making forward and reverse mutagenic primers an By making forward and reverse mutagenic primers and using other primers that anneal to common vector sequences, two PCR rd using other primers that anneal to common vector sequences, two PCR reactions are carried out to amplify 5’- and 3’-portions of the DNA to be meactions are carried out to amplify 5’- and 3’-portions of the DNA to be mutated. The two PCR products are mixed and used for another PCR using tutated. The two PCR products are mixed and used for another PCR using the outer primers only. Part of this product is then suncloned to replace the he outer primers only. Part of this product is then suncloned to replace the region to be mutated in the starting molecule.region to be mutated in the starting molecule.

Fig. 1. Unidirectional deletion Fig. 1. Unidirectional deletion

mutagenesismutagenesis

Fig. 2. PCR mutagenesis. X is the mutated site in the Fig. 2. PCR mutagenesis. X is the mutated site in the PCR productPCR product

J6 APPLICATIONS OF J6 APPLICATIONS OF CLONINGCLONING

ApplicationsApplications The various applications of gene The various applications of gene cloning incluse recombinant protein production, cloning incluse recombinant protein production, genetically modified organism, DNA fingerpringenetically modified organism, DNA fingerprinting, diagnostic kits and gene therapy.ting, diagnostic kits and gene therapy.

Recombinant proteinRecombinant protein By inserting the gene fo By inserting the gene for a rare protein into a plasmid and expressing it r a rare protein into a plasmid and expressing it in bacteria, large amounts of recombinant proteiin bacteria, large amounts of recombinant protein can be produced. If post-translational modificn can be produced. If post-translational modifications are critical, the gene may have to be exprations are critical, the gene may have to be expressed in aeukaryotic cell.essed in aeukaryotic cell.

Genetically modified organismsGenetically modified organisms Imtroducting a foreign gene into Imtroducting a foreign gene into an organism which can propagate creats a genetically modified organ organism which can propagate creats a genetically modified organism. Transgenic sheep have been created to produce foreign protanism. Transgenic sheep have been created to produce foreign proteins in their milk.eins in their milk.

DNA fingerprintingDNA fingerprinting Hybridizing Southern blots of genomic DNA Hybridizing Southern blots of genomic DNA with probes that recognize simple nucleotide repeats gives a patterwith probes that recognize simple nucleotide repeats gives a pattern that is unique to an individual and can be used as a fingerprint. Tn that is unique to an individual and can be used as a fingerprint. This has applications in forensic science, animal and plant breeding his has applications in forensic science, animal and plant breeding and evolutionary studies.and evolutionary studies.

Medical diagnosisMedical diagnosis The sequence information derived from clonin The sequence information derived from cloning medically important genes has allowed the design of many diagnig medically important genes has allowed the design of many diagnistic test kits which can help predict and confirm a wide range of disstic test kits which can help predict and confirm a wide range of disorders.orders.

Gene therapyGene therapy Attempts to correct a genetic disorder by delivering Attempts to correct a genetic disorder by delivering a gene to patient are described as gene therapy.a gene to patient are described as gene therapy.

Fig. 1. DNA fingerprinting showing how two VNTR alleles might be inheFig. 1. DNA fingerprinting showing how two VNTR alleles might be inherited (see text). (a) Parental VNTR alleles. (b) Agarose gel analysis of VNrited (see text). (a) Parental VNTR alleles. (b) Agarose gel analysis of VN

TR alleles.TR alleles.