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BIOLOGY 2C03 (150)
Joe Kim (16)
Lecture 7

Lecture 7 - February 14, 2013.docx

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McMaster University
Joe Kim

BIO 2C03 2013 Lecture 7 – February 14, 2013 Mapping Human Genes  Need high density maps o Problem – too few genes have been identified (phenotypically) to create a genetic map o Solution – use a genetic locus other than a gene – DNA markers or nucleotide polymorphisms  DNA marker is locus with a known location that comes in identifiable variations (alleles) in a population  Nucleotide polymorphisms can be detected using molecular techniques  These allelic variations segregate according to Mendelian principles and show linkage relationships  Polymorphism – alternative forms of a genetic locus that differ in terms of nucleotide sequence o Locus – a position on the length of DNA and is not necessarily a gene o DNA/molecular marker – polymorphism useful for mapping o Very common; members of a single species show enormous amounts of sequence variation Molecular Phenotypes  Alleles of DNA markers are detected by molecular tools including PCR (polymerase chain reaction) or Southern blot o Refer to observations as molecular phenotypes  The frequency of DNA polymorphisms in the human genome is high (1/350 base pairs, bp)  Classes of polymorphisms 1. Single nucleotide polymorphism (SNPs) – a difference in a single nucleotide at a DNA locus  Eg/ Two alternate alleles for this DNA sequence  In a population, there may be many alternate alleles  Detect by southern blot analysis, PCR analysis, Direct sequencing, or Allele-specific oligonucleotide (ASO) hybridization 2. Microsatellites or short tandem repeats (STRs) 3. Minisatellites or variable number tandem repeats (VNTRs) Restriction Fragment Length Polymorphism (RFLP) Analysis  Restriction Fragment Length Polymorphism (RFLP)– restriction enzymes cut double-stranded DNA at specific sequences Running DNA Fragments on a gel RFLP Genotyping = determining genotype How RFLP Works 1 BIO 2C03 2013  These changes are useful for mapping if the population is polymorphic (many alleles in the population) o Monomorphic – no variation in population (>99% of individuals in population have same sequence)  Can use molecular markers in linkage mapping 7+9 = 16 recombinants RF = 16/100 x 100% RF = 16% Or a map distance of 16 mu 2 BIO 2C03 2013  RFLP analysis is used to follow disease-causing alleles  Assumption – a polymorphic marker is very close to the gene – it is unlikely that crossover will occur  Need to find an allele of the marker that is linked to the disease-causing allele in the population  Eg/ Sickle Cell Anemia – Kan and Dozy, 1978 – looked for polymorphisms that were frequent in each group of distinct genotypes o Hb /Hb = homozygous for wildtype allele o Hb /Hb = heterozygous carriers o Hb /Hb = homozygous for Sickle Cell Anemia  Obtaining Samples  The 13 kb fragment (marker) segregates with the Hb allele 3 BIO 2C03 2013 Microsatellites/Short Tandem Repeats (STR)/Simply Sequence Repeats  Useful and ubiquitous molecular marker  2-6 bp that are repeated in tandem (in a row) form a few to hundreds of times  Eg/ 2 bp repeats – (GTn ; n = number of times the repeat appears in an allele  Eg/ 3 bp repeats – (CAGn – a higher number of these repeats in a gene is often associated with disease  Eg/ 6 bp repeats – found at telomeres  Detection of microsatellites o PCR amplification and gel electrophoresis  PCR primers are in a unique sequence, not repeats marker / marker x marker /marker 10  6 10 marker /marker  Detection of microsatellite polymorphisms using PCR: 4 BIO 2C03 2013
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