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23 Cards in this Set
- Front
- Back
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Biotechnology
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the commerical application of recombinant DNA technology
applications in: medical diagnostic production of medicines and vaccines chemical production genetically modified food products (plants and animals) criminal investigations |
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Generation of Herbicide Resistance in Plants
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Glyphosate: herbicide that is effective at low concentrations: inhibits an essential chloroplast enzyme
Fig 19-1 |
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Genetically Engineered Rice: golden rice
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has enhance levels of B-Carotene- a vitamin A precursor
Express thee genes that encode B-carotene biosynthetic enzymes |
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Insulin Production in E. coli
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Insulin was the first protein to be manufactured using rDNA in 1982: consists of 2 subunits: alpha (21 aa) and beta (30 aa)
IN the original process the gene for each subunit was expressed separately as a "fusion protein" with B-galactosidase Fusion proteins were expressed and isolated from culture and the proteins were cleaved from the B-Gal by treating with cyanogen bromide Insulin subunits were purified and mixed to form active insulin. |
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Synthesis of Recombinant Human Insulin in E.coli
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Fig 19.3
transform into E.coli host Extract and purify B-gal/Insulin fusion proteins Threat with cynogen bromide to cleave A and B chains Purify, mix A and B chains to form functional insulin. |
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Table 19.1 Some Genetically Engineered Pharm Products now Available or under Development
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Tissue plasminogen activator tPA (heart attacks)
Human Growth Hormone (dwarfism) Monoclonal Abs against vascular endothelial growth factor (cancers) Human clotting factor VIII |
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Edible Vaccine products in Plants
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Expression of an antigenic subunit of Hep B virus in tobacco
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Production of Edible Vaccine in Plants
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Gene from a human pathogen is inserted into a vector taken up by a bacterium that infects plants
Bacteria infect potato leaf segments Leaf segments sprout in to whole plants carrying gene from human pathogen Eating raw potato triggers immune response to pathogen |
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Diagnosis and Screening for Genetic Disorders
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Aminocentesis and chorionic villus sampling (CVS), cell samples from unborn child can be retrieved for analysis:
count chromosomes (e.g. downs syndrome- 3 copies of chr 21) |
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Use of Recombinant DNA Technology to Diagnose Sickle Cell Anemia
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RFLP Analysis
e.g. Sickle cell anemia A to T transversion causes loss of an MstII restriction site so one large restriction fragment is seen instead of the usual two on a southern blot. |
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Use of Allele-Specific Oligos in Disease Diagnosis
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Allele SPecific Oligonucleotides (ASO) used when there is no change in a restriction enzyme site:
can detect a single nucleotide difference useful when the mutation does not affect a restriction enzyme site. |
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Microarrays: Revolutionizing Diagnostics and Treatments
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1. Small oligonucleotides are attached to tiny areas on a glass slide: appx 12,000 genes on one slide
2. DNA isolated from cells and regions of selected genes amplified by PCR and labeled with a fluorescent dye. 3. These are hybridized with DNA chip and strength of binding measured by strength of signal that binds 4. Using oligonucs on chip with mismatches that correspond w/mutations in gene: patient DNA sample can be scanned for mutations in many genes: p53 gene and BRCA1 |
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DNA Microarray Analysis
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mRNA present only in normal cells (a)
mRNA present only in cancer cells (at other end) mRNA present in equal amts in normal in cancer cells in the middle |
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Gene Expression Profiling of Lymphoid Malignancies
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Diagnosis of cancer is based largely on morphological examination of the tumor sample coupled w/clinical date
However, the treatment responses of patients w/in a diagnostic category tend to be very heterogenous A particular type of cancer in two different people may appear to be similar, but at the molecular level they are likely very different: molecular heterogeneity among tumors accounts for the majority of the variability of responses to treatments better means of diagnosing tumors and placing them into homogenous subtypes are needed. |
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Molecular Diagnosis of Lymphoid Malignancies
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1. Has the potential to guide patients to the most appropriate treatment regimen for their cancer
2. Provide a blueprint of abnormal molecular activity of the cancer cell: this can lead to the development of molecularly targeted terapies that have both specificity and potency for defined cancer types molecular definition of a cancer type: all cases w/in a particular type should be homogenous |
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Gene Expression Profiling of Lymphoid Malignancies: Lymphochip
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using Lymphocip DNA microarray: contains >18,500 cDNAs (genes) derived from normal germinal center B cells: mature B cells
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Diffuse Large B cell Lymphoma (DLBCL)
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Form of Non-Hodgkins Lymphoma
At least 2 distinct forms (subtypes) of DLBCL Subtypes cannot be distinguished based on morphology, clinical data, immunohistochemistry, or genetics Multi-agent chemotherapy used to treat these cases (CHOP) Cures 40% of patients Remainder eventually succumb to the disease. |
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Gene Expression Profiling of Lymphoid Malignancies: Lymphochip + DLBCL
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Lymphochip microarray analysis was performed on tumor samples from patients w/DLBCL and the gene expression profiles from these samples was compared
Results subdivided DLBCL into 2 subgroups based on the expression profile of about 100 genes Sub 1: Germinal center B- like DLBCL, Closely resembles normal germinal center B cells in gene expression profile Sub 2: Lack expression of germinal center B cell genes and expressed genes characteristic of activated B cells. |
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What is the significance of the Lymphochip/DLBCL findings?
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Sub 1: Germinal center B, favorable prognosis, about 75% cured w/current chemotherapeutic treatment
Sub 2: activated B center, poor prognosis, less than 25% respond to current treatment and achieve long-term remission demonstrates that genomic-scale gene expression analysis can define clinically important subtypes of human cancers by understanding the different signaling pathway that are active in different subtypes of cancers, new therapeutics can be designed to specifically inhibit those pathways. |
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Gene Therapy
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Insert a permanent copy of a normal gene into an affected patient: gene will produce a normal protein product that can perform the function that the mutant copy cannot: permanent-not in germ-line (not inherited)
Main challenge is in gene delivery: viral vectors first successful gene therapy performed on a patient with SCID: caused by mutation in adenine deaminase (ADA) gene ADA is involved in detoxification of excessive amts of adenosine and adenosine analogs |
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Table 19.2 Vectors for Gene Therapy
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Adenovirus vector; for lung resp tract
Retroviruses vector: for proliferating cells etc. |
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Treatment of SCID using Gene Therapy
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Bacterium carrying plasmid w/cloned normal human ADA gene + genetically disabled retrovirus
Cloned ADA gene is incorporated into virus Retrovirus infects blood cells, transfers ADA gene to cells Cells are grown in culture to ensure ADA gene is active Genetically altered cells are re-implanted, produce ADA |
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Gene Therapy Trials and Vectors
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Cancer (63%)
Infectious diseases (6.4%) Retrovirus (34.1%) Adenovirus (26.9%) Naked/plasmid DNA (11%) |
