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Genetic and Physical Mapping Lecture 2
Why map genes?  Many diseases are partially genetic – Also: environmental factors, randomness 2.	We want to identify these genes – Early diagnosis for abortion or regular 	checks 	– First step towards developing treatment 3.  Individual sequencing is too costly (today) – Sequence a small number of markers 	– Analyze statistically via biological principles
[object Object]
based on the use of genetic techniques to construct linkage maps showing the positions of genes and other sequence features in a genome
Physical mapping:
uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the exact position of genes and other sequences,[object Object]
Genetic Linkage Maps A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker Markers can be expressed DNA regions (genes) or DNA segments that have no known coding function but whose inheritance pattern can be followed. DNA sequence differences are especially useful markers because they are plentiful and easy to characterize precisely Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist among individuals so that they are detectable among different members in family studies Polymorphisms are variations in DNA sequence that occur on average once every 300 to 500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility
Stages of Mapping a Gene • Demonstrate disease is hereditary 	– Show it runs in families • Linkage analysis to identify region 	– Widely-spaced markers, e.g. RFLPs • Association analysis to narrow region 	– Closely-spaced markers, usually SNPs • Clone the gene within found region 	– Investigate its metabolic relevance
Summary A genetic linkage map shows the relative locations of specific DNA markers along the chromosome 2. Five major DNA markers for genetic mapping:  ,[object Object]
RAPD
SNP
AFLP
Microsatellites3. A physical map shows the exact position of genes and other sequences on the chromosome 4. Five physical mapping methods:  ,[object Object]
fingerprinting
 chromosome walking,
STS and FISH ,[object Object]
Types of  DNA Markers 1. Restriction fragment length polymorphisms (RFLP) 2. RAPD-Random amplified polymorphic DNA (RAPD) 3. Microsatellite, simple sequence repeat (SSR) markers or short tandem repeat (STR) 4. Single nucleotide polymorphisms (SNPs) 5. Amplified fragment length polymorphism (AFLP)
[object Object]
Minisatellites
Variable number of tandem repeats = VNTR
High number of alleles
High level of heterozygocity
Problem: southern blots and radioactive probes, minisatellites are to long to be amplified by PCR
Not evenly spread over the genome
Microsatellites
Single Nucleotide Polymorphisms (SNP),[object Object]
Minisatellites
Microsatellites
Standard tools for mapping studies and linkega analysis
Mostly (CA)n repeats
Tri-en tetranucleotide repeats replace more and more the dinucleotide repeats, because of the better results
Dinucleotide-repeats are volnurable to “replication slippage” during PCR as such that each allel can give a small ladder on a gel
Development of multiplex PCR reactions
Single Nucleotide Polymorphisms (SNP),[object Object]
Physical Mapping ,[object Object],[object Object]
Physical Map of a Chromosome Contig:  ,[object Object],Depicts genetic markers and DNA sequences between the markers measured in base pairs (High Resolution)
Physical Mapping Methods Optical mapping 2. Restriction fragment fingerprinting 3. Chromosome walking 4. Sequence tagged site (STS) mapping 5. Fluorescent in situ hybridization (FISH)
2 Restriction fragment fingerprinting ,[object Object]
The digested DNA is labeled with radioactive or fluorescent dye and run on a sequencing gel
The fingerprint data is collected and analyzed for contigassemblyDisadvantages: labor intensive and difficult to fill gaps.

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Lecture 2

  • 1. Genetic and Physical Mapping Lecture 2
  • 2. Why map genes? Many diseases are partially genetic – Also: environmental factors, randomness 2. We want to identify these genes – Early diagnosis for abortion or regular checks – First step towards developing treatment 3. Individual sequencing is too costly (today) – Sequence a small number of markers – Analyze statistically via biological principles
  • 3.
  • 4. based on the use of genetic techniques to construct linkage maps showing the positions of genes and other sequence features in a genome
  • 6.
  • 7.
  • 8.
  • 9.
  • 10. Genetic Linkage Maps A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker Markers can be expressed DNA regions (genes) or DNA segments that have no known coding function but whose inheritance pattern can be followed. DNA sequence differences are especially useful markers because they are plentiful and easy to characterize precisely Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist among individuals so that they are detectable among different members in family studies Polymorphisms are variations in DNA sequence that occur on average once every 300 to 500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility
  • 11. Stages of Mapping a Gene • Demonstrate disease is hereditary – Show it runs in families • Linkage analysis to identify region – Widely-spaced markers, e.g. RFLPs • Association analysis to narrow region – Closely-spaced markers, usually SNPs • Clone the gene within found region – Investigate its metabolic relevance
  • 12.
  • 13.
  • 14.
  • 15. RAPD
  • 16. SNP
  • 17. AFLP
  • 18.
  • 21.
  • 22.
  • 23.
  • 24. Types of DNA Markers 1. Restriction fragment length polymorphisms (RFLP) 2. RAPD-Random amplified polymorphic DNA (RAPD) 3. Microsatellite, simple sequence repeat (SSR) markers or short tandem repeat (STR) 4. Single nucleotide polymorphisms (SNPs) 5. Amplified fragment length polymorphism (AFLP)
  • 25.
  • 26.
  • 28. Variable number of tandem repeats = VNTR
  • 29. High number of alleles
  • 30. High level of heterozygocity
  • 31. Problem: southern blots and radioactive probes, minisatellites are to long to be amplified by PCR
  • 32. Not evenly spread over the genome
  • 34.
  • 37. Standard tools for mapping studies and linkega analysis
  • 39. Tri-en tetranucleotide repeats replace more and more the dinucleotide repeats, because of the better results
  • 40. Dinucleotide-repeats are volnurable to “replication slippage” during PCR as such that each allel can give a small ladder on a gel
  • 41. Development of multiplex PCR reactions
  • 42.
  • 43.
  • 44.
  • 45.
  • 46.
  • 47.
  • 48.
  • 49.
  • 50. Physical Mapping Methods Optical mapping 2. Restriction fragment fingerprinting 3. Chromosome walking 4. Sequence tagged site (STS) mapping 5. Fluorescent in situ hybridization (FISH)
  • 51.
  • 52.
  • 53. The digested DNA is labeled with radioactive or fluorescent dye and run on a sequencing gel
  • 54. The fingerprint data is collected and analyzed for contigassemblyDisadvantages: labor intensive and difficult to fill gaps.
  • 55.
  • 56.
  • 57.
  • 58.
  • 59. Clones hybridizing with the same single copy marker are considered to be overlapping
  • 60.
  • 61.
  • 62.
  • 63.
  • 64. PCR confirmation of STS markers in the genome Each STS contains a unique sequence
  • 65.
  • 66.
  • 67.
  • 68.
  • 69. Probes specific to chromosome regions 1p34– 35 and 1p36 were labeled using the ULYSIS Oregon Green 488 (U21659) and Alexa Fluor 594 (U21654) Nucleic Acid Labeling Kits, respectively.(http://www.probes.com/servlets/photohigh?fileid=g001276)
  • 70. FISH mapping of BACs (bacterial artificial chromosomes)
  • 71. Application of FISH in physical mapping Jackson S. et al. 2000. Genetics, 156: 833-838
  • 72.
  • 73.
  • 74.
  • 75.
  • 76.
  • 77.
  • 78.
  • 79. Mice,
  • 82.
  • 83. A dozen other bacteria have been completely sequenced and all their genes identified, although the functions of most are unknown.
  • 84. Major progress has been made in mapping human genes, and a "rough draft" of the human genome was read by 2000.
  • 85.