Molecular mapping

molecular mapping

1. M OLECULAR Mapping By Usman Arshad

2. Contents Genetic mapping: Virtual or relational mapping Physical mapping: systematic analysis Chromosome walking: find a gene on chromosome New techniques for mapping .

3. Why map before sequencing? Major problem in large-scale sequencing: Current technologies can only sequence 600–800 bases at a time. We need to sequence 30 billion bp in order to perfectly sequence human genome One solution: make a physical map of overlapping DNA fragments: Top-Down approach Chromosomal libraries: 46 chromosomes/23 pairs Genomic library for many fragments from each chromosome Determine sequence of each fragment Then assemble to form contiguous sequence

4. Mapping I Mapping is identifying relationships between genes on chromosomes Just as a road map shows relationships between towns on highway: fine maps Two types of mapping: genetic and physical

5. Mapping II Genetic mapping Based on differences in recombination frequency between genetic loci: meiosis Physical mapping Based on actual distances in base pairs between specific sequences found on the chromosome Most powerful when genetic and physical mapping are combined

6. Genetic mapping Based on recombination frequencies The further away two points are on a chromosome, the more recombination there is between them Because recombination frequencies vary along a chromosome, we can obtain a relative position for the loci Distance between the markers

7. Genetic mapping Genetic mapping requires that a cross be performed between two related organisms The organism should have phenotypic differences (contrasting characters like red and white or tall and short etc) resulting from allele differences at two or more loci The frequency of recombination is determined by counting the F 2 progeny with each phenotype

8. Genetic mapping example I Genes on two different chromosomes Independent assortment during meiosis (Mendel) No linkage Dihybrid ratio F 1 9 : 3 : 3 : 1 F 2 P

9. Genetic mapping example II Genes very close together on same chromosome Will usually end up together after meiosis Tightly linked F 1 1 : 2 : 1 F 2 P

10. Genetic mapping example III Genes on same chromosome, but not very close together Recombination will occur Frequency of recombination proportional to distance between genes Measured in centiMorgans =cM Recombinants Non-parental features One map unit = one centimorgan ( cM ) = 1% recombination between loci

11. cM or centimorgan 1% Recombination = 1 cM

12. Genetic markers Genetic mapping between positions on chromosomes Positions can be genes Responsible for phenotype Examples: eye color or disease trait: limited Positions can be physical markers DNA sequence variation

13. Physical markers Physical markers are DNA sequences that vary between two related genomes Referred to as a DNA polymorphism Usually not in a gene Examples RFLP SSLP SNP

14. RFLP Restriction-fragment length polymorphism Cut genomic DNA from two individuals with restriction enzyme Run Southern blot Probe with different pieces of DNA Sequence difference creates different band pattern GGATCC CCTAGG GTATCC GATAGG GGATCC CCTAGG 200 400 GGATCC CCTAGG GCATCC GGTAGG GGATCC CCTAGG 200 400 * * 200 400 600 1 2 * * 2 1

15. SSLP/Microsatellites Simple-sequence length polymorphism Most genomes contain repeats of three or four nucleotides Length of repeat varies due to slippage in replication Use PCR with primers external to the repeat region On gel, see difference in length of amplified fragment ATCCTAC GACGACGACGATT GATGCT 12 18 1 2 2 1 ATCCTAC GACGACGACGACGACGATT GATGCT

16. SNP Single-nucleotide polymorphism One-nucleotide difference in sequence of two organisms Found by sequencing Example: Between any two humans, on average one SNP every 1,000 base pairs ATCGATTGCCATGAC ATCGATGGCCATGAC 2 1 SNP

17. Physical mapping Determination of physical distance between two points on chromosome Distance in base pairs Example: between physical marker and a gene Need overlapping fragments of DNA Requires vectors that accommodate large inserts Examples: cosmids, YACs, and BACs

18. Molecular mapping Digest DNA Electrophorese - + Southern blot Hybridize with probe

19. Physical Mapping Systems (like a Filing system of clones) Yeast Artificial Chromosomes (YACs) 200-1000 kb Bacteriophage P1 90 kb Cosmids 40 kb Bacteriophage l 9-23 kb Plasmids (2-6 kb)

20. Large insert vectors Lambda phage Insert size: 20–30 kb Cosmids Insert size: 35–45 kb BACs and PACs (bacterial and P1 artificial chromosomes (Viral) respectively) Insert size: 100–300 kb YACs (yeast artificial chromosomes) Insert size: 200–1,000 kb

21. large-insert vectors Lambda phage and cosmids Inserts stable But insert size too small for large-scale sequencing projects YACs Largest insert size But difficult to work with due to instability

22. BACs and PACs BACs and PACs Most commonly used vectors for large-scale sequencing Good compromise between insert size and ease of use Growth and isolation similar to that for plasmids

23. Contigs Contigs are groups of overlapping pieces of chromosomal DNA Make contig uous clones For sequencing one wants to create “minimum tiling path” Contig of smallest number of inserts that covers a region of the chromosome genomic DNA contig minimum tiling path

24. Contigs from overlapping restriction fragments Cut inserts with restriction enzyme Look for similar pattern of restriction fragments Known as “fingerprinting” Line up overlapping fragments Continue until a contig is built

25. Restriction mapping applied to large-insert clones Generates a large number of fragments Requires high-resolution separation of fragments Can be done with gel electrophoresis

26. Analysis of restriction fragments Computer programs perform automatic fragment-size matching Possibilities for errors Fragments of similar size may in fact be different sequences Repetitive elements give same sizes, but from different chromosomal locations

27. Gel image processing © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

28. FPC: fingerprint analysis window © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

29. Building contigs by probing with end fragments Isolate DNA from both ends of insert and mix Label and probe genomic library Identify hybridizing clones Repeat with ends of overlapping clones

30. Chromosome walking Combines probing with insert ends and restriction mapping First find hybridizing clones Then create a restriction map Identify the clone with the shortest overlap Make probe from its end Repeat process probe library probe library

31. Sequence separation Terminated chains need to be separated Requires one-base-pair resolution See difference between chain of X and X+1 base pairs Gel electrophoresis Very thin gel High voltage Works with radioactive or fluorescent labels A T C G – +

32. Capillary electrophoresis Newer automated sequencers use very thin capillary tubes Run all four fluorescently tagged reactions in same capillary Can have 96 capillaries running at the same time 96–well plate robotic arm and syringe 96 glass capillaries load bar

33. Sequence reading of radioactively labeled reactions Radioactively labeled reactions Gel dried Placed on X-ray film Sequence read from bottom up Each lane is a different base – + C A G T C A G T

34. Sequence reading of fluorescently labeled reactions Fluorescently labeled reactions scanned by laser as a particular point is passed Color picked up by detector Output sent directly to computer

35. Optical Mapping Single-molecule technique Individual DNA molecules attached to glass support Restriction enzymes on glass are activated When DNA is cut, microscope records length of resulting fragments Has potential to rapidly generate restriction maps

36. Summary Basics of mapping Genetic mapping Based on recombination frequencies Physical mapping Requires overlapping DNA fragments Can use restriction enzymes Probing with end fragments Combination: chromosome walking

37. THANKS