Section J Analysis and application of cloning DNA

1. Molecular Biology Section J Analysis and application of cloning DNA

2. Molecular Biology Section J -Analysis and application of cloning DNA • J1 CHARACTERIZATION OF CLONES • J2 NUCLEIC AND SEQUENCING • J3 POLYMERASE CHAIN REACTION • J4 ORGANIZATION OF CLONED GENES • J5 MUTAGENESIS OF CLONED GENE • J6 APPLICATIONS OF CLONING

3. Molecular Biology J1 CHARACTERIZATION OF CLONES • Characterization Determining various properties of recombinant DNA molecule, such as size, restriction map, orientation of any gene present and nucleotide sequence, constitutes the process of clone characterization. It requires a purified preparation of the cloned DNA. • Restriction mapping Digesting recombinant DNA molecules with restriction enzymes, alone and in combination, allows the construction of a diagram(restriction map) of the molecules indicating the cleavage position and fragment sizes. • Partial digestion The partial digestion of end-labeled DNA fragments with restriction enzymes, and sizing of fragments produced, also enables a restriction map to be constructed.

4. Molecular Biology • Labelilg nucleic acid DNA and RNA can be end-labeled using polynucleotide kinase or terminal transferase. Uniform labeling requires polymerases to synthesize a complete labeled strand. • Southern and Northern blotting The nucleic acid in lanes of a gel is transferred to a memebrane, bound and then hybridized with a labeled nucleic acid probe. Washing removes nonhybridized probe, and the membrane is then treated to reveal the bands produced. Specific RNA species are detected on Northern blots, whereas the DNA bands on Southern blots could be genes in genomic DNA or parts of cloned genes.

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

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

7. Molecular Biology Fig. 3. Southern blotting.

8. Molecular Biology J2 NUCLEIC AND SEQUENCING • DNA sequencing The two main methods of DNA sequencing are Maxam and Gilbert chemical method in which end-labeled DNA is subjected to base-specific cleave reaction prior to gel separation, and Snger’s enzymic method. The latter uses dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer. • RNA sequencing A set of four Rnase that cleave 3’ to specific nucleotides are used to produce a ladder of fragments from end-labeled RNA. Polyacrylamide gel electrophoresis analysis allows the sequence to be read.

9. Molecular Biology • Sequence databasesNewly determined DNA ,RNA and protein sequences are entered into databases(EMBL and GeneBank). These collections of all known sequences are available for analysis by computer. • Analysis of sequencesSpecial computer software is used to search nucleic acid and protein sequences for the presence of or similarities • Genome sequencing projectsThe entire genome sequences of several organisms have been determined and those of other organisms are in progress. Often a genetic map is first produced to aid the project.

10. Molecular Biology Fig. 1. DNA sequencing. (a) An example of a Maxam and Gilbert sequencing gel; (b) Sanger sequencing.

11. Molecular Biology J3 POLYMERASE CHAIN REACTION • The PCR cycleThe reaction cycle comprises a 95℃ step to denature the duplex DNA, an annealing step of around 55℃ to allow the primers to bind and a 72℃ polymerization step. Mg2+ and dNTP are required in addition to template, primers, buffer and enzyme. • TemplateAlmost any source that contains one or more intact target DNA molecule can, in theory, be amplified by PCR, providing appropriate primers can be designed. • PrimersA pair of oligonucleotides of about 18-30 nt with similar G+C content will serve as PCR primers as long as they direct DNA synthesis towards one another. Primers with some degeneracy can also be used if the target DNA sequence is not completely known.

12. Molecular Biology • EnzymesThermostable DNA polymerases(e.g. Taq polymerase) are used in PCR as they survive the hot denaturation step. Some are more error-prone than others. • PCR optimizationIt may be necessary to vary the annealing temperature and/or the Mg2+ concentration to obtain faithful amplification. From complex mixtures, a second pair of nested primers can improve specificity. • PCR variationVariation on basic PCR include quantitative PCR, degenerate oligonucleotide primer PCR(DOP-PCR), inverse PCR, multiplex PCR, rapid amplification of cDNA ends(RACE) and PCR mutagenesis.

13. Molecular Biology 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 amplified (molecules 2 and 7). After a few more cycles these become the major product.

14. Molecular Biology J4 ORGANIZATION OF CLONED GENES • OrganizationThe polarity of oligo(dT)-primed cDNA clones is often apparent 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 clone is not obvious, but can be determined by mapping and probing experiments. • Mapping cDNA on genomic DNASouthern blotting, using probes from part of a cDNA clone, can show which parts of a genomic clone have corresponding sequences. • S1 nuclease mappingThe 5’- or 3’- of a transcript can be identified by hybridizing a longer, end-labeled antisense fragment to the RNA. The hybrid is treated with nuclease S1 to remove single-stranded regions, and remaining fragment’s size is measured on a gel. • Primer extensionA primer is extended by a polymerase until the end of the template is reached and the polymerase dissociates. The length of the extended product indicates the 5’-end of the template.

15. Molecular Biology • Gel retardationMixing a protein extract with a labeled DNA fragment and running the mixture on a native gel will show the presence of DNA-protein complexes as retarded bands on the gel. • Dnase I footprintingThe ‘footprint’ of a protein bound specifically to a DNA sequence can be visualized by treating the mixture of end-labeled DNA plus protein with small amounts of Dnase I prior to running the mixture on a gel. The footprint is a region with few bands in a ladder of cleave products. • Reporter genesTo verify the function of promoter, it can be joined to the coding region of an easily detected gene(reporter gene) and the protein product assayed under condition when the promoter should be active.

16. Molecular Biology Fig. 1. S1 nuclease mapping the 5′-end of an RNA. * = position of end label. RNA 5’ 3’ DNA 3’ * 5’ Add S1 nuclease RNA 5’ 3’ DNA 3’ 5’ PAGE Analysis

17. Molecular Biology Fig. 2. Primer extension. * = position of end label.

18. Molecular Biology J5 MUTAGENESIS OF CLONED GENE • Deletion mutagenesisProgressively deleting DNA from one end is very useful for defining the important of particular sequences. Unidirectional deletion 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 nuclease then creates blunt end molecules for ligation, and transformation generates then deleted clones. • Site-directed mutagenesisChanging one or a few nucleotides at a particular site usually involves annealing a mutagenic primer to a template followed by complementary strand synthesis by a DNA polymerase. Formerly. Single-stranded templates prepared using M13 were used, but polymerase chain reaction(PCR) techniques are now preferred. • PCR mutagenesisBy making forward and reverse mutagenic primers and 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 mutated. 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 region to be mutated in the starting molecule.

19. Molecular Biology Fig. 1. Unidirectional deletion mutagenesis

20. Molecular Biology Fig. 2. PCR mutagenesis. X is the mutated site in the PCR product

21. Molecular Biology J6 APPLICATIONS OF CLONING • ApplicationsThe various applications of gene cloning incluse recombinant protein production, genetically modified organism, DNA fingerprinting, diagnostic kits and gene therapy. • Recombinant proteinBy inserting the gene for a rare protein into a plasmid and expressing it in bacteria, large amounts of recombinant protein can be produced. If post-translational modifications are critical, the gene may have to be expressed in aeukaryotic cell.

22. Molecular Biology • Genetically modified organismsImtroducting a foreign gene into an organism which can propagate creats a genetically modified organism. Transgenic sheep have been created to produce foreign proteins in their milk. • DNA fingerprintingHybridizing Southern blots of genomic DNA with probes that recognize simple nucleotide repeats gives a pattern that is unique to an individual and can be used as a fingerprint. This has applications in forensic science, animal and plant breeding and evolutionary studies. • Medical diagnosisThe sequence information derived from cloning medically important genes has allowed the design of many diagnistic test kits which can help predict and confirm a wide range of disorders. • Gene therapyAttempts to correct a genetic disorder by delivering a gene to patient are described as gene therapy.

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