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19 Cards in this Set

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