1. Map Based Cloning (Positional cloning)
Cloning New gene based on already developed genetic map information

2. Cloning of new Gene Cloning strategies Diverse on the situation
1. ortholog analysis
2. candidate gene approach
3. comparing difference in mRNA
4. using protein information
5. mutants analysis
6. map based cloning-positional cloning

3. 그림2

4. Starting point
From phenotype
From mutant
From DNA infomation

5. Mutation analysis Random mutation transposon tagging- knock down
chemical & physical mutagen
insertional mutagenesis
transposon tagging- knock down
- activation tagging
TDNA tagging-plant

6. Rarely, loss-of-function mutations are dominant.
Dominant-negative mutations – alleles of a gene encoding subunits of multimers that block the activity of subunits produced by normal alleles

7. Gain-of-function mutations are almost always dominant.
Rare mutations that enhance a protein function or even confer a new activity on a protein
Antennapedia is a neomorphic mutation causing ectopic expression of a leg-determining gene in structures that normally produce antennae.

8. Mutations in genes encoding the molecules that implement expression may affect transcription, mRNA splicing, or translation.
Usually lethal
Mutations in tRNA genes can suppress mutations in protein-coding genes.
Nonsense suppressor tRNAs

9. Nonsense suppression
(a) Nonsense mutation that causes incomplete nonfunctional polypeptide
(b) Nonsense-suppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide.
Figure 8.28
Fig. 8.32

10. Classical cloning strategy –from protein information
A pedigree of the royal family descended from Queen Victoria In which hemophilia A is segregating
Figure a
Fig a

11. Blood-clotting cascade in which vessel damage causes a cascade of inactive factors to be converted to active factors
Figure b

12. Blood tests determine if active form of each factor in the cascade is present.
Figure c

13. Techniques used to purify Factor VIII and clone the gene
Figure d

14. Positional Cloning – Step 1
Find extended families in which disease is segregating.
Use panel of polymorphic markers spaced at 10 cM intervals across all chromosomes.-interval mapping
300 markers total
Determine genotype for all individuals in families for each DNA marker.
Look for linkage between a marker and disease phenotype.-fine mapping

15. Once region of chromosome is identified, a high resolution mapping is performed with additional markers to narrow down region where gene may lie.
Figure 11.17

16. Positional cloning – Step 2 identifying candidate genes
Once region of chromosome has been narrowed down by linkage analysis to 1000 kb or less, all genes within are identified.
Candidate genes
Usually about 17 genes per 1000 kb fragment
Identify coding regions
Computational analysis to identify conserved sequences between species
Computational analysis to identify exon-like sequences by looking for codon usage, ORFs, and splice sites
Appearance on one or more EST clones derived from cDNA

17. Computational analysis of genomic sequences to identify candidate genes
Figure 11.19

18. Gene expression patterns can pinpoint candidate genes.
Look in public database of EST sequences representing certain tissues.
Northern blot
RNA transcripts in the cells of a particular tissue (e.g., with disease) separated by electrophoresis and probed with candidate gene sequence

19. Northern blot example showing SRY candidate for testes determining factor is expressed in testes, but not lung, ovary, or kidney.
Figure 11.20
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20. Positional cloning – Step 3
Find the gene responsible for the phenotype.
Expression patterns
RNA expression assayed by Northern blot or PCR amplification of cDNA with primers specific to candidate transcript
Look for misexpression (no expression, underexpression, overexpression).
Sequence differences
Missense mutations identified by sequencing coding region of candidate gene from normal and abnormal individuals
Transgenic modification of phenotype
Insert the mutant gene into a model organism.

21. Example: Positional Cloning of Cystic Fibrosis Gene
Linkage analysis places CF on chromosome 7
Figure a

22. Northern blot analysis reveals only one of candidate genes is expressed in lungs and pancreas.
Figure b

23. Location and number of mutations indicated under diagram of chromosome
Every CF patient has a mutated allele of the DFTR gene on both chromosome 7 homologs.
Figure c
Location and number of mutations indicated under diagram of chromosome

24. CFTR is a membrane protein. TMD-1 and TMD-2 are transmembrane domains.
Figure d

25. Proving CFTR is the right gene
Phenotype eliminates gene function.
Cannot use transgenic technology
Instead perform CRFT gene “knockout” in mouse to examine phenotype without CRFT gene.
Targeted mutagenesis
Introduce mutant CFTR into mouse embryonic cells in culture.
Rare double recombinant events with homologous wild-type CFTR gene are selected for.
Mutant cell is introduced into normal mouse embryos where they incorporate into germ line.
Knockout mouse created

26. Transgenic analysis can prove candidate gene is disease locus.
Figure 11.21

27. Genetic dissection of complex traits
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28. Incomplete penetrance – when a mutant genotype does not always cause a mutant phenotype
No environmental factor associated with likelihood of breast cancer
Positional cloning identified BRAC1 as one gene causing breast cancer.
Only 66% of women who carry BRAC1 mutation develop breast cancer by age 55.
Incomplete penetrance hampers linkage mapping and positional cloning.
Solution – exclude all non-disease individuals from analysis
Requires many more families for study
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29. Variable expressivity
Expression of a mutant trait differs from person to person.
Include any degree of mutant phenotype as evidence for presence of mutant allele.
Phenocopy
Disease phenotype is not caused by any inherited predisposing mutation.
Decreases power to detect correlation between inheritance of disease locus and expression of the disease
Genetic heterogeneity
Mutations at more than one locus cause same phenotype.
Multiple families used in most studies
If different families have different gene mutations, power of statistic to detect linkage will drop significantly.
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30. Polygenic inheritance
Two or more genes interact in the expression of phenotype.
QTLs, or quantitative trait loci
Unlimited number of transmission patterns for QTLs
Discrete traits – penetrance may increase with number of mutant loci
Expressivity may vary with number of loci.
etc.
Many other factors complicate analysis.
Some mutant genes may have large effect.
Mutations at some loci may be recessive while others are dominant or codominant.
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31. Identifying contributing loci for complex traits
Analyze two contrasting phenotypes from two inbred mouse lines.
Eliminates heterogeneity
Only two alternative alleles at every marker
No environmental differences
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32. Pulp content in tomato – example of complex trait method
Identification of two inbred strains with extreme, reproducible differences in pulp content
Cross plants from high- and low-pulp strains.
Cross identical F1 hybrids to generate several hundred F2 offspring. Range of phenotypes produced
Figure b
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Fig b

33. Identifying regions of chromosome where pulp content genes may lie
Determine genotype at polymorphic DNA markers spaced at 20 cM intervals in each F2 plant.
Look for correlation between marker genotype and pulp phenotype.
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35. Haplotype association studies for high-resolution mapping in humans
Haplotypes are sets of closely linked alleles.
Specific combination of two or more DNA marker alleles situated close together on the same DNA molecule
Usually SNPs
Distance between SNPs on a haplotype must be short enough that they stay associated during transmission over many generations.
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36. Formation of haplotypes over evolutionary time
Figure 11.25
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37. Ancient disease loci are associated with haplotypes.
Start with population genetically isolated for a long time such as Icelanders or Amish.
Collect DNA samples from subgroup with disease.
Also collect from equal number of people without disease.
Genotype each individual in subgroups for haplotypes throughout entire genome.
Look for association between haplotype and disease phenotype.
Association represents linkage disequilibrium.
If successful, it provides high resolution to narrow parts of chromosomes.
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38. Haplotype analysis provides high resolution gene mapping.
Figure 11.26
Fig
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39. 혈압·맥박·엉덩이크기도 ‘대물림’ 국내연구진, 해당 유전자 6개 세계 첫 발견
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40. Integration of linkage, physical, and sequence maps
Provides check on the correct order of each map against other two
SSR and SNP DNA linkage markers readily integrated into physical map by PCR analysis across insert clones in physical map
SSR, SNP (linkage maps), and STS markers (physical maps) have unique sequences 20 bp or more, allowing placement on sequence map.
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41. Conserved segments of syntenic blocks in human and mouse genomes
Figure 10.12
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Fig

42. Social, ethical, and legal issues
Privacy of genetic information
Limitations on genetic testing
Patenting of DNA sequences
Society’s view of older people
Training of physicians
Human genetic engineering
Somatic gene therapy – inserting replacement genes
Germ-line therapy – modifications of human germ line
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43. New approach to studying biological systems has made possible:
Systems Biology – the global study of multiple components of biological systems and their interactions
New approach to studying biological systems has made possible:
Sequencing genomes
High-throughput platform development
Development of powerful computational tools
The use of model organisms
Comparative genomics
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