Chapter 9: Applications of Recombinant DNA Technology

1. Chapter 9: Applications of Recombinant DNA Technology Linnea Fletcher Ph.D. BIOL 2316

2. Site-Specific Mutagenesis Describe the practical application of site-directed mutagenesis, and why this technique is more valuable than classical non-specific mutagenesis. Describe how PCR can be used to quickly generate a site-specific mutation in a gene. Describe the procedures used to create a �knock-out� mouse with a site-specific mutation in a gene. Why must knockout mice be interbred before the effects of the knockout mutation can be known? What are some human diseases that have been modeled in knockout mice?

3. Figure 9.1 An example of site-specific mutagenesis using PCR. Figure 9.1 An example of site-specific mutagenesis using PCR.

4. Analysis of DNA Polymorphisms What is the difference between a mutation, an allele, and a DNA polymorphism? What is the approximate frequency of DNA polymorphisms in the human genome? Why are the majority of DNA polymorphisms found in noncoding regions of the genome? Make a table listing 3 classes of DNA polymorphisms, how these classes differ from each other, and their relative frequency in the human genome. Why are cSNPs of special interest to medical research? Describe how SNPs can be detected by RFLP analysis. Describe how SNPs can be detected by ASO hybridization analysis. What advantages does this approach offer? Describe how SNPs can be detected by DNA microarray analysis. What advantages does this approach offer?

5. Figure 9.2 Southern blot analysis method for studying SNPs that affect restriction sites. A 7-kb section of the chromosome has BamHI sites at each end. SNP allele 1 (top) has a BamHI site 2 kb from the left end, whereas SNP allele 2 (bottom) has a CG base pair in place of a TA base pair, so that the BamHI site has been lost. BamHI digestion with DNA samples from individuals with different SNP genotypes, followed by Southern blot analysis using the probe shown, gives the DNA banding patterns at the bottom. Figure 9.2 Southern blot analysis method for studying SNPs that affect restriction sites. A 7-kb section of the chromosome has BamHI sites at each end. SNP allele 1 (top) has a BamHI site 2 kb from the left end, whereas SNP allele 2 (bottom) has a CG base pair in place of a TA base pair, so that the BamHI site has been lost. BamHI digestion with DNA samples from individuals with different SNP genotypes, followed by Southern blot analysis using the probe shown, gives the DNA banding patterns at the bottom.

6. Figure 9.3 PCR method for studying SNPs that affect restriction sites. A 2,000-bp section of the chromosome has SNP alleles 500 bp from the left end. The TA-to-CG change from SNP allele 1 (top) to SNP allele 2 (bottom) alters a BamHI site to a sequence that is not recognized by a restriction enzyme. PCR of DNA samples from individuals with different SNP allele genotypes using the left and right primers shown, followed by BamHI digestion, gives the DNA banding pattern at the bottom. Figure 9.3 PCR method for studying SNPs that affect restriction sites. A 2,000-bp section of the chromosome has SNP alleles 500 bp from the left end. The TA-to-CG change from SNP allele 1 (top) to SNP allele 2 (bottom) alters a BamHI site to a sequence that is not recognized by a restriction enzyme. PCR of DNA samples from individuals with different SNP allele genotypes using the left and right primers shown, followed by BamHI digestion, gives the DNA banding pattern at the bottom.

7. Figure 9.4 Typing of an SNP by oligonucleotide hybridization analysis. An oligonucleotide that is completely complementary to the normal allele is hybridized to the target DNA under conditions that favor a perfect match between probe and target. If hybridization occurs, the target DNA has the normal allele, but if hybridization does not occur, the target DNA has a base mismatch�that is, an SNP polymorphism. Figure 9.4 Typing of an SNP by oligonucleotide hybridization analysis. An oligonucleotide that is completely complementary to the normal allele is hybridized to the target DNA under conditions that favor a perfect match between probe and target. If hybridization occurs, the target DNA has the normal allele, but if hybridization does not occur, the target DNA has a base mismatch�that is, an SNP polymorphism.

8. Figure 9.5 Illustration of an experiment using a DNA microarray. Figure 9.5 Illustration of an experiment using a DNA microarray.

9. STRs and VNTRs What are some other terms used to describe a STR? What is the most common way that STR analysis is performed? What are some practical applications of STR analysis? What is another term used to describe a VNTR? How are VNTR analyzed? Why is PCR not used for VNTR analysis? What are 2 types of VNTR?

10. Figure 9.6 Using PCR to determine which STR (microsatellite) alleles are present. Genomic DNA is isolated, and PCR primers flanking an STR locus are used to amplify the repeats. The sizes of the DNA fragments produced are determined by agarose gel electrophoresis. In the figure, STR allele 1 has 6 repeats of GATA, and STR allele 2 has 10 repeats of GATA. The gel shows the three possible genotypes for these two alleles: (6,6) [i.e., both homologues have the six-repeat allele], (10,10), and (6,10). In reality, there is likely to be a lot of variation in repeat number at an STR locus. Figure 9.6 Using PCR to determine which STR (microsatellite) alleles are present. Genomic DNA is isolated, and PCR primers flanking an STR locus are used to amplify the repeats. The sizes of the DNA fragments produced are determined by agarose gel electrophoresis. In the figure, STR allele 1 has 6 repeats of GATA, and STR allele 2 has 10 repeats of GATA. The gel shows the three possible genotypes for these two alleles: (6,6) [i.e., both homologues have the six-repeat allele], (10,10), and (6,10). In reality, there is likely to be a lot of variation in repeat number at an STR locus.

11. DNA Molecular Testing for Human Disease Mutations What is genetic testing? DNA molecular testing? What are some of the complications of genetic testing? How genetic testing distinct from screening for a disease? From a diagnostic test? What are the three main reasons it is done? What is the function of normal alleles of BRCA1 and BRCA2, and what are clinical signs of loss of function mutations in these genes? Why is there no single DNA molecular test for BRCA gene mutations? Why is a BRCA lesion not diagnostic for a cancerous tumor? What are some examples of human genetic diseases that can be tested for by RFLP analysis?

12. Figure 9.7 The beginning of the ?-globin gene, mRNA, and polypeptide showing the normal Hb-A sequences and the mutant Hb-S sequences. The sequence differences between Hb-A and Hb-S are shown in bold. The mutation alters a DdeI site (boxed in the Hb-A DNA). Figure 9.7 The beginning of the ?-globin gene, mRNA, and polypeptide showing the normal Hb-A sequences and the mutant Hb-S sequences. The sequence differences between Hb-A and Hb-S are shown in bold. The mutation alters a DdeI site (boxed in the Hb-A DNA).



15. Figure 9.8 Detection of sickle-cell gene by the DdeI restriction fragment length polymorphism. (a) DNA segments showing the DdeI restriction sites. (b) Results of analysis of DNA cut with DdeI, subjected to gel electrophoresis, blotted, and probed with a ?-globin probe.Figure 9.8 Detection of sickle-cell gene by the DdeI restriction fragment length polymorphism. (a) DNA segments showing the DdeI restriction sites. (b) Results of analysis of DNA cut with DdeI, subjected to gel electrophoresis, blotted, and probed with a ?-globin probe.

16. Figure 9.8 Detection of sickle-cell gene by the DdeI restriction fragment length polymorphism. (a) DNA segments showing the DdeI restriction sites. (b) Results of analysis of DNA cut with DdeI, subjected to gel electrophoresis, blotted, and probed with a ?-globin probe. Figure 9.8 Detection of sickle-cell gene by the DdeI restriction fragment length polymorphism. (a) DNA segments showing the DdeI restriction sites. (b) Results of analysis of DNA cut with DdeI, subjected to gel electrophoresis, blotted, and probed with a ?-globin probe.



19. Using PCR Approaches Describe how the point mutation of the GLC1A gene that gives rise to open-angle glaucoma is tested by ASO hybridization. Why are the PCR products first run out on an electrophoresis gel prior to blotting to a membrane? How can the homozygotes be distinguished from the heterozygotes? What are some advantages of reverse ASO hybridization for genetic testing? Why is multiplex PCR required for some reverse ASO hybridization tests?

20. Figure 9.9 DNA molecular testing for mutations of the open-angle glaucoma gene GLC1A, using PCR and allele-specific oligonucleotide (ASO) hybridization. (a) Sequence of part of the GLC1A gene from a heterozygote showing a mutation from C to T, causing a Pro-to-Leu change in the polypeptide at amino acid 370. (b) Sequences of the two allele-specific oligonucleotides (ASOs), one for the wild-type allele and one for the mutant allele. (c) Results (theoretical) of hybridization with radioactive ASOs for homozygous normal, homozygous mutant, and heterozygous individuals. Figure 9.9 DNA molecular testing for mutations of the open-angle glaucoma gene GLC1A, using PCR and allele-specific oligonucleotide (ASO) hybridization. (a) Sequence of part of the GLC1A gene from a heterozygote showing a mutation from C to T, causing a Pro-to-Leu change in the polypeptide at amino acid 370. (b) Sequences of the two allele-specific oligonucleotides (ASOs), one for the wild-type allele and one for the mutant allele. (c) Results (theoretical) of hybridization with radioactive ASOs for homozygous normal, homozygous mutant, and heterozygous individuals.

21. Isolation of Human Genes What is positional cloning and what was the first human genetic disease whose gene was cloned this way? How long did it take to locate the cystic fibrosis gene in this way? How could other species be used to help to identify which of the clones contain the cystic fibrosis gene, and not noncoding DNA? How could northern blots be used to help to identify which of the clones contain the cystic fibrosis gene, and not noncoding DNA? Why was cultured normal sweat gland cells used to generate a cDNA library for screening with a cystic fibrosis sequence probe? Once a candidate gene was discovered, how was this gene verified as the cystic fibrosis defect? How many different mutations have been identified for cystic fibrosis to date?

22. Figure 9.10 Chromosome walking. (From Biochemistry, by Donald Voet and Judith G. Voet. Copyright � 1990 Donald Voet and Judith G. Voet. Reprinted by permission of John Wiley & Sons, Inc.) Figure 9.10 Chromosome walking. (From Biochemistry, by Donald Voet and Judith G. Voet. Copyright � 1990 Donald Voet and Judith G. Voet. Reprinted by permission of John Wiley & Sons, Inc.)

23. DNA Typing What are some other terms for DNA fingerprinting? What individuals share a DNA fingerprint? Why is it more difficult to establish a positive identity than it is to exclude an individual by DNA fingerprinting? How are inaccuracies avoided in DNA fingerprinting? How many people has the Innocence Project exonerated for crimes by 2004, using DNA typing of crime evidence? What are some other applications, other than paternity and forensics investigations, for DNA typing? What are some examples of fossil DNA that have been analyzed?

24. Figure 9.11 DNA typing to determine paternity. Figure 9.11 DNA typing to determine paternity.






30. Figure 9.12 Sir Alec Jeffreys, the discoverer of VNTRs. He is holding examples of DNA fingerprints. Figure 9.12 Sir Alec Jeffreys, the discoverer of VNTRs. He is holding examples of DNA fingerprints.

31. Gene Expression How can hybridization techniques be used to assay the level of gene expression in an organism? How can RT-PCR be used to assay the level of gene expression, and how can this technique determine conditions where the message RNA is alternatively spiced? Describe how the yeast two-hybrid system assays protein-protein interactions, and how this can be used to discover the molecular basis of human disease.

32. Figure 9.13 Regulation of transcription of the yeast GAL1 gene by glucose. Glucose was added at time zero, and the amount of GAL1 transcribed was analyzed at various times thereafter by blotting and probing, as described in the text. (From Figure 5, Johnston, M., Flick, J. S., and Pexton, T., 1994. Multiple mechanisms provide rapid and stringent glucose repression of GAL gene expression in Saccharomyces cerevisiae. Mol Cell Biol 14:3834�3841.) Figure 9.13 Regulation of transcription of the yeast GAL1 gene by glucose. Glucose was added at time zero, and the amount of GAL1 transcribed was analyzed at various times thereafter by blotting and probing, as described in the text. (From Figure 5, Johnston, M., Flick, J. S., and Pexton, T., 1994. Multiple mechanisms provide rapid and stringent glucose repression of GAL gene expression in Saccharomyces cerevisiae. Mol Cell Biol 14:3834�3841.)

33. Figure 9.14 Alternative sex-specific pre-mRNA splicing in the fru (fruitless) gene of Drosophila melanogaster. A section of the fru pre-mRNA transcript is shown. In males and females, different 5� splice sites 1,590 nt apart are used with the same 3� splice site; the result is a smaller mRNA in the male. Figure 9.14 Alternative sex-specific pre-mRNA splicing in the fru (fruitless) gene of Drosophila melanogaster. A section of the fru pre-mRNA transcript is shown. In males and females, different 5� splice sites 1,590 nt apart are used with the same 3� splice site; the result is a smaller mRNA in the male.

34. Gene Therapy What is an advantage of germ-line gene therapy, compared to somatic cell gene therapy? What vector do most gene therapies use in order to boost the transfection efficiency? What are some possible deleterious effects of transfection on a transgenic cell? The first successful gene therapy trial was in 1990 when children with SCID were given an ADA replacement gene. Why do you think that this particular disease is more amenable than most genetic disease to successful gene replacement therapy, and how successful was this therapy, as it turns out? How successful has gene therapy been in the treatment of sickle cell anemia?

35. Commercial Products Animals have been used for the production of biopharmaceuticals (referred to as �pharm animals�). What are some reasons that expression is often targeted in mammary tissue? What are some reasons that pharm animals are preferred to microbial fermentations for production of biopharmaceuticals, especially the injectable types?

36. Figure 9.16 Production of a recombinant protein product (here, the protein encoded by the gene of interest, GOI) in a transgenic mammal�in this case, a sheep. Figure 9.16 Production of a recombinant protein product (here, the protein encoded by the gene of interest, GOI) in a transgenic mammal�in this case, a sheep.

37. Plant Products Describe how Agrobacterium tumifasciens can be used to introduce genes into plant cells. How can monocots be transformed with recombinant DNA? Describe how Roundup-ready crops are engineered and how they boost agricultural productivity. Describe how antisense mRNA is used to increase the shelf life of Flavr Savr tomatoes. Describe how potatoes have been engineered to produce hepatitis B vaccine. What advantage could such an approach be for third-world countries?

38. Figure 9.17 Formation of tumors (crown galls) in plants by infection with certain species of Agrobacterium. Tumors are induced by the Ti plasmid, which is carried by the bacterium and integrates some of its DNA (the T, or transforming, DNA) into the plant cell�s chromosome. Figure 9.17 Formation of tumors (crown galls) in plants by infection with certain species of Agrobacterium. Tumors are induced by the Ti plasmid, which is carried by the bacterium and integrates some of its DNA (the T, or transforming, DNA) into the plant cell�s chromosome.

39. Figure 9.18 Making a transgenic, RoundupTM-tolerant tobacco plant by introducing a modified form of the bacterial gene for the enzyme EPSPS that is resistant to the herbicide. The gene encoding the bacterial EPSPS was spliced to a petunia sequence encoding a transit peptide for directing polypeptides into the chloroplast, and the modified gene was inserted into a T-DNA vector and introduced into tobacco by Agrobacterium-based transformation. Both the native and the modified bacterial EPSPS are transported into the chloroplast. When plants are sprayed with Roundup, wild-type plants die because only the native chloroplast EPSPS is present, and it is sensitive to the herbicide, but the transgenic plants live because they contain the bacterial EPSPS that is resistant to the herbicide. Figure 9.18 Making a transgenic, RoundupTM-tolerant tobacco plant by introducing a modified form of the bacterial gene for the enzyme EPSPS that is resistant to the herbicide. The gene encoding the bacterial EPSPS was spliced to a petunia sequence encoding a transit peptide for directing polypeptides into the chloroplast, and the modified gene was inserted into a T-DNA vector and introduced into tobacco by Agrobacterium-based transformation. Both the native and the modified bacterial EPSPS are transported into the chloroplast. When plants are sprayed with Roundup, wild-type plants die because only the native chloroplast EPSPS is present, and it is sensitive to the herbicide, but the transgenic plants live because they contain the bacterial EPSPS that is resistant to the herbicide.

40. Extra credit and practice-each one is 5 points! 9.11 9.16 9.24

Chapter 9: Applications of Recombinant DNA Technology

Chapter 9: Applications of Recombinant DNA Technology

Presentation Transcript

Table of Contents – pages iv-v

Recombinant DNA Technology

Recombinant DNA and Genetic Engineering

Recombinant DNA Technology

Chapter 9 Biotechnology and Recombinant DNA

Recombinant DNA Technology

Outline

Recombinant DNA Technology

Recombinant DNA Technology

Recombinant DNA Technology

Chapter 8: Recombinant DNA Technology and Molecular Cloning

Chapter 19 – Molecular Genetic Analysis and Biotechnology

DNA recombinant technology & genetic engineering (1) several concepts

Recombinant DNA Technology

Chapter 20

重组 DNA 技术 Recombinant DNA Technology

Recombinant DNA I

DNA Recombinant Technology

DNA Technology and Genomics

Biotechnology

Medical biotechnology Recombinant protein based pharmaceuticals and diagnostics

Ch. 13.1: BIOTECHNOLOGY

Chapter 9: Applications of Recombinant DNA Technology