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19 Cards in this Set
- Front
- Back
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What is the Goal of Reverse Genetics?
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To identify a mutant gene that causes a monogenic disease with unknown altered proteins.
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In general terms, what are the 5 steps in Reverse Genetics?
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1. Determine chromosome on which disease gene is located
2. Find disease gene on that Ch' 3. Search for expressed DNA sequences w/in the gene region 4. Isolate cDNA corresponding to a disease gene candidate 5. Sequence cDNA of normal and affected individuals that matches the cDNA isolated in Step 4 to ID a mutation |
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What is the ultimate goal of Reverse Genetics?
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To correct the disease using eukaryotic expression vectors
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What 2 steps are required for isolating the chromosome containing a disease gene?
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1. Establish linkage between the disease gene and a marker (RFLP)
2. Map the RFLP to a specific chromosome by using FISH |
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How can you establish a linkage relationship for a Disease Gene and marker?
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By using the genomic DNA of an AFFECTED FAMILY - many members.
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What technique is used for establishing linkage?
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Southern Blot - use restriction endonuclease to cleave DNA, compare fragments between family members w/ disease vs. those without
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How is the linkage relationship determined once a gene is linked to an RFLP/marker?
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By looking at several generations to establish the number of recombinants
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What does using FISH do for chromosome determination?
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-Use DNA probe specific for RFLP
-Plate w/ all chromosomes -Chromosome w/ disease gene should light up |
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Once the chromosome has been established, how can you find the exact disease gene?
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Look for other RFLPs closer to the disease gene.
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After getting as close as possible to disease gene w/ RFLPs, then what?
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Chromosomal walking
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What are the steps in Chromosomal walking?
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1. Construct Probe from known 5' flanking RFLP sequence
2. Use Probe to find a Clone from a genomic library 3. Use 3' end of clone as a probe to find a new clone from genomic library ...continue until the clone contains the 3' flanking RFLP |
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What is achieved by Chromosomal walking?
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You know the successive, partially overlapping short fragments of DNA within the section of Ch' containing disease gene.
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How do you determine which of these fragments is or contains the disease gene?
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By searching for expressed DNA sequences - cDNA
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What is cDNA?
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DNA only containing exon-encoded information - made from mRNA.
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How do you go about searching for cDNA?
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1. Make probes from clones obtained in Ch' walking
2. Hybridize probes to mRNA from diseased tissues - Northern blot |
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What does the Northern blot w/ clone DNA probes vs. disease RNA tell you?
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If they hybridize, you know the protein is expressed in disease.
-This is a disease gene candidate! |
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What can be done with the gene candidate?
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mRNA that hybridized to probe can be used to make cDNA via Reverse Transcriptase
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What does knowing the cDNA from the candidate disease gene allow?
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If the same is sequenced from populations of normal and affected individuals, identification of the mutation.
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Once the candidate disease gene's mutation is identified what is the goal?
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To correct the mutation with eukaryotic expression vectors.
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