L54-55: Molecular Medicine I-II Flashcards
Explain use of antibodies in ELISAs and Western Blotting
1.) ELISAs: - Used to detect and quantify antigens in vitro. - Two types: sandwich (used routinely for detection of PTH, HbA1c and CRP) and indirect (used for diagnosis of HIV through viral core protein and autoimmune diseases) - Sandwich: coat well with antibody, add serum, antigen specific to antibody binds, add enzyme-linked specific antibody, add substrate and develop color or fluorescence - Indirect: Antigen is attached to reaction well, well filled with sample and antibodies contained in the sample bind to the antigen, second reporter-linked antibody reactive against other antibodies is added 2.) Western Blotting - Technique that uses specific antibodies to indicate the molecular weight of a protein - Technique: protein extracts are subject to electrophoresis, proteins transferred to membrane, incubated with specific antibody, antibody binds and identifies protein of interest. *Elisa reveals quantitative info about an antigen, whereas a Western blot can discriminate between isoforms of an antigen (based on MW)
Describe 4 methods of gene therapy
1.) Viral gene delivery: viruses are used to insert genetic material into host’s genome. Harmful part of viruses genome is removed, replaced with gene of interest. This technique relies of viruses ability to efficiently integrate info into host chromosomes. 2.) Non-viral methods: DNA delivered into cells via liposomes, naked DNA, complexed DNA and artificial chromosomes. 3.) Ex vivo gene therapy: removal of pts cells, manipulation of cells in lab, reimplantation of modified cells 4.) In vivo gene therapy: aim of treating cells directly in the patient and avoiding process of harvesting and re-implanting cells. Works best on epithelial cells that are exposed on surface of tissue and accessible to gene vector. This utilizes viral gene delivery systems.
Describe principles of and methods for gene therapy
1.) Principles: mutation to be cured must be recessive (adding functional copy of gene will remedy the defect). Cells in question must be accessible to manipulation. Gene therapy in this case would be somatic gene therapy and not germ-line therapy. 2.) Methods: viral gene delivery, non-viral methods of gene delivery, ex vivo gene therapy, in vivo gene therapy
Describe the use of antibodies in the therapy of cancer
- CA cells present often with abnormal surface glycoproteins. Antibodies can be produced that bind to these surface markers. Bind of such antibodies identifies a cell as malignant. They can be used alone or conjugated to anticancer drugs, toxins or radioactive molecules to kill the CA cells - Breast cancer: Herceptin is monoclonal antibody that binds erbB-2 recetors, which inhibits proliferation of tumors. Trastuzumab-DM is a drug that is herceptin conjugated with chemotherapeutic agent.
Compare and contrast direct sequencing and indirect marker analysis methods for the detection of mutations. Potential problems with each
- Direct: if exact mutation is known, one can test pts DNA for it. Problem: pt might have novel, rare unknown mutation that is not detected - Indirect: if exact mutation is not known, one looks for presence of linked marker. Problem: marker might have separated from disease allele
With the following techniques, what sample is used and what is detected? Direct sequencing, WES, SNP typing, PCR, rtPCR, ASO, gene expression array, methylated DNA PCR, comparative genome hybridization, FISH, ELISA and Western blot
Compare and contrast WGS, WES ad SNP typing
- WGS = whole genome sequencing. Entire genome is 3 billion basepairs in length. Difficult to sequence, not feasible or clinically necessary as only 30 million basepairs actually codes for exon sequences. - WES = whole exome sequencing. 1% of genome codes for exon sequences. This is an affordable technique to characterize the most relevant fraction of the human genome. - SNP typing = When comparing genome of individuals, there are fewer than 10 million single nucleotide polymorphisms in the genome. SNP typing provides this information. In analysis of this, only a few thousand SNPs actually matter in diseases.
Describe prenatal diagnosis
Two methods of prenatal diagnoses are possible 1.) Amniocentesis (gold standard): utilizes amniotic fluid from 15-16 weeks gestation and analyzes fetal skin cells 2.) CVS: utilizes cells from chorion taken from 10-12 weeks.
Explain PCR and its application in the analyses of polymorphisms and gene expression
1.) PCR - Amplification of DNA framents. Mutation can be detected with oliogonucleotides that exclusively bind to either mutant or wildtype alleles. Insertions or delections between primer bindings site become immediately obvious because they lead ot a variation of length of amplified fragment - AFLP 2.) rtPCR - Quantification of transcript by detecting the amount of amplified DNA. Used for detection of retroviral load in blood samples and expression of oncogenes and tumor suppressor genes in cancer cells.
Explain the potential applications of pharmacogenetics. Provide examples
- Pharmacogenetics correlates genotypes and individual drug responses that result from genetic polymorphisms 1.) Malignant hyperthermia: rare autosomal disorder that is leading cause of death during anesthesia. Halothane and succinyl choline trigger calcium release in muscle cells and heat generated from pumping calcium back into SR causes hyperthermia. This is due to a mutation in ryanodine receptor. 2.) Warfarin drug response: Warfarin acts by inhibiting vitamin K epoxide reductase. Polymorphism in VKORC1 affects sensitivity. Polymorphism in CYP2C9, which encodes cytochrome P450, leads to slow metabolism of warfarin and therefore leads to a higher sensitivity to drug.
Describe the limits of DNA sequencing
- Sequencing will show if a person carries a mutation in the heterozygous or homozygous form. Does not show on which chromosome the mutation is or even if it is paternal or maternal. Sequencing will not show if a person has a deletion or a duplication of a chromosomal region.
Describe production of polyclonal and monoclonal antibodies
- Polyclonal antibodies: antibodies against different epitopes of an antigen. Protein of interest is purified, injected into animal, collecting serum from animal and extracting antibodies against protein - Monoclonal antibodies: antibodies against one specific epitope . Animal is injected with antigen, collect B-lymphocyte fraction from spleen of animal and fuses the cells to myeloma cell line, resulting hybridoma cells are immortal and produce antibodies. Next step, one hybridoma cell line is selected from many diff clones by quality and specificity of antibodies it produces. This cell line is then grown in culture almost indefinitely and continues to produce only one specific antibody
Explain the importance of copy number variation and two techniques to detect it
- CNV describes observation that chromosomal regions can be present in non-diploid states, ie. you can have more or less than two copies of a gene. - Techniques: - 1.) Comparative Genome Hybridization: use samples of single stranded DNA from pt and a normal control that are labeled with fluorescent dye. Then mix and allow hybridization with chromosomes in metaphase. CNV is evident by uneven labeling via fluorescence. - 2.) FISH: identification of chromosomal locus on a metaphase chromosome by hybridization with a specific fluorescent probe. Then count fluorescent markers.
Compare and contrast Allele Specific Oligonucleotide (ASO) arrays and gene expression arrays
- ASOs are a tool to detect SNPs and mutations. They utilize short primers (20 nucleotides) that hybridize with their complementary base pairs. DNA-microarrays involve thousands of oligonucleotide spots printed on very small areas. This can then be used as diagnostic tool to do genetic screening on a pt for all known mutant alleles. Can be used with low amounts of pts DNA since amplification step proceeds it - Gene expression arrays: oligonucleotides can be used for large-scale analysis of gene expression. Array is made from oligonucleotides that each bind to just one specific gene transcript. This allows for quantification of thousands of mRNAs in one study. Evaluation of level of expression of transcripts can be deduced from these arrays – no expression, overexpression or underexpression
Contrast somatic and germ line gene therapies
- Somatic: repairing genome of differentiated cells of body. This is what is done in gene therapy. - Germ-line: repairing/altering genome of reproductive cells thus permanently altering the genetic composition of an individual and its offspring. This is prohibited.
