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Markers and Maps.docx

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Bhagwati Gupta

October 28 , 2013 Biology 2C03: Genetics Markers and Maps Mapping - The relative location of genes or loci along a chromosome - Map units are proportional to base pairs - Maps are collinear - Types of maps which are related (collinear)  Linkage maps  Represents linkage distance  Cytological map:  Polygene chromosome  Salivary gland in drosophila – replicate many times but chromosomes do not segregate  Bands = condensation more dense areas  Puffs = more accessible regions to factors  Banding pattern is stereotypic and fix  Physical map:  Actual bases of DNA that you would obtain by sequencing the chromosome  Distance between the genes measured by the number of bases - Relationship is unique to the organism in which map units are proportional to the base pairs – distances are not the same in other organism - Genome map: comes from linkage, cytological, and physical maps - in situ can also give us chromosome maps  Labeling DNA and hybridize with the DNA you are studying to locate genes that correspond to the probes  Does not give information of gene localization Mapping Human Genes - Recombination analysis of X-linked genes (limited application) - Cannot subject humans to experimentation - Little information available for distance between human genes - Recombination that segregates the two markers are the most important in a cross - Recombination is rare but does occur Pedigrees for Haemophilia and Colourblindness 1. A women heteroxygous for haemophilia A and colourblindness, 3% chance of her son having only one condition  RF = 3%, amp distance 3 m.u.  Determined by lod (logarithm of odds) score – pooled data from a number of pedigrees 2. Green weakness gene and haemophilia A  Map distance = 8 m.u. 3. Haemophilia B and colourblindness (may appear unlinked in a small sample)  Map distance = 36 m.u. - Related to the distance on the chromosome - Genetic linkage map and cytological map - Recombination increase as we move to the arms - Centromeres are more tight and are not as free to crossover Mapping Human Genes - High density maps  Problem: too few genes have been identified (phenotypically) to create a genetic map  Solution: use another genetic marker = DNA markers or nucleotide polymorphisms - DNA marker is a piece of DNA of known size that comes in identifiable variations (alleles) - Molecular markers are loci in the genome whose sequences can be detected using molecular techniques - These allelic variations segregate according to Mendelian principles and show linkage relationships What is a Polymorphism? - Alternative forms of a chromosomal locus that differ in terms of nucleotide sequence - We can refer to a locus as simply a region on DNA and not necessarily a gene - If a polymorphism is useful for mapping, it is called a DNA marker or a molecular marker - How common are polymorphisms?  Very, members of a single species show an enormous amounts of sequence variation - Some polymorphisms are not useful - Alleles of DNA markers are detected by molecular tools including PCR (polymerase chain reaction) and Southern blot. We might refer to the observations as molecular phenotypes - The frequency of DNA polymorphisms in the human genome is high, about 1/350 base pairs (bp) - Classes of polymorphisms: 1. Single nucleotide polymorphisms (SNPs) – including those digested by REs (snipSNPs) 2. Microsatellites or short tandem repeats (STRs)  Repeats in the bases  Very short in length 3. Minisatellites or variable number tandem repeats (VNTRs)  Sequences repeated many times on different chromosomes  No specific protein that is coded  No strong selection pressure so the number and size is variable from generation to generation 4. Insertion and deletions (indels): short and long  Small sequences that are deleted or randomly inserted Single Nucleotide Polymorphism (SNPs) - A difference in a single nucleotide at a DNA locus - These are two alternate alleles for this sequence of DNA - In a population, there may be many alternate alleles - Detecting SNPs:  Southern Blot analysis, PCR analysis, direct sequencing or allele- specific oligonucleotide(ASO) hybridization October 29 , 2013 Visualizing DNA Fragments - Restriction enzymes cut double-stranded DNA at specific sequences  Useful for polymorphisms  Blunt ends (Pvu II) or  Complementary sticky ends with complementary base parings (HindIII)  Enzymes are very specific to the DNA sequences - Running DNA fragments on a gel  Standard technique to detect DNA based on size Locating Specific DNA Sequences Using a Complementary Probe - Southern blotting: take a membrane and place on top of the gel, placed in a dish and using a weighted and an electric charge, the DNA binds to the membrane and apply a probe - Visualize specific DNA fragments - A probe is a single stranded DNA sequence that is: 1) Labeled 2) Complementary to the sequence of a unique region of the genome snipsSNPs - SNPs that produce different DNA fragments upon digestion - There are two alternate alleles produce different DNA fragments when digested with an enzyme such as PstI - Detecting snipSNPs: carry out PCR, digest DNA with RE 9e.g., PstI), and run agarose gel to see DNA bands - Revealed by an enzyme So what is a RFLP or Retriction Fragment Length Polymorphism? - Different individuals have different nucleotide sequences (especially in noncoding DNA) resulting in different patterns of restriction fragments - A SNP can change the nucleotide sequence to remove a restriction enzyme site or add one - Monomorphic: no variation in population (>99% of individuals I population have the same sequence)  Both chromosomes have similar banding patterns - Polymorphic: variation in population  Banding pattern of the chromosomes is different We can use these Molecular Markers in Linkage Mapping - Heterozygote will have a combination of the bands of both homozygotes - Homozygous pairings – heterozygous offspring
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