BIOL 1411 1st Edition Lecture 29Outline of Last Lecture I. MutationsOutline of Current Lecture I. Genetic Analysis II. Relating Genotype with phenotypeLecture- Costs and Benefits of Mutationso Costs Some germ line and somatic cell mutations are harmful or lethal, leading to genetic diseases and cancer Mutations commonly responsible for recessive mendelian traits occurred long ago, and exists as genetic loado Benefits Provide the raw material for evolution in the form of genetic diversity Evolution would not be possible without mutation- Genetic markers and the discovery of Disease-causing alleleso Genetic markers provide reference leci for associating genotype with phenotype.o Knowledge of two mutations is needed: One influences the disease phenotype and the other is a “marker mutation” Co-inheritance of the marker of the marker and the disease- causing allele occurs due to genetic linkage; i.e. the loci are closely linked - Polymerase Chain Reactiono Copies of DNA sequences can be made by the polymerase chain reaction (PCR) techniqueo PCR is a cyclical process: DNA fragments are denatured by heating Primers, plus dNTPs and DNA polymerase are added New DNA strands are synthesized o A cycle of steps Features of each PCR cycle (30-35 total)- Step 1: 95C; separated (denatured) the two strands of the DNA helix to generate single-strand templateThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Step 2: (50-60 degrees C) Primer base pairs (anneal) to target sites- Step 3” 72 C; heat tolerant polymerase synthesizes DNA from 3’-OH of DNA primer- Product of the polymerase chain reactiono PCR generates many copies of the DNA fragment- referred to as amplifying the sequence. Copies of target region double each cycleo Complementary primers, about 15-30 bases long, are synthesized (IDT) accordingto known sequence- Types of DNA sequence variationo Two types of common genetic variation (polymorphisms in DNA sequence): Single nucleotide polymorphism (SNPs): inherited variations involving a single base- originating by point mutations Short tandem repeats (STRs): short repetitive sequences occurring side byside on chromosomes, usually in noncoding regions - Genetic Markerso Polymorphisms are used as genetic markers in analyses of humans and other organisms Sequence differences (alleles) in genetic markers must be identifiable by current DNA analysis methods; co-dominance is best Restriction enzymes can be used to identify SNPs (and insertions and deletions) in restriction sites. SNPs can be directly assayed STRs vary in length and there are multiple alleles at each marker locus- Gel Electrophoresiso DNA fragments can be separated by sixe using gel electrophoresiso A mixture of fragments in placed in a well of a semisolid gel. An electric field is applied across the gel negatively charged DNA fragments move towards positive endo Smaller fragments move through the gel faster than larger ones - Restriction fragment length polymorphisms (RFLPs)o Restrictions enzymes cut DNA at specific sequences generating smaller fragmentso Mutations at restriction sites the change the ability of the enzyme to cut can be assayed as restriction fragment length polymorphismso RFLPs are observed as bands on an electrophoresis gel following digestion of PCR-amplified target or hybridization of digested DNA with probe - Direct Screening of SNPs o Single nucleotide polymorphisms can be detected directly using the allele-specific oligonucleotide hybridization methodo Short synthetic DNA strands called oligonucleotide probes hybridize with denatured PCR productso Hybridization to the DNA containing the complementary SNP is detected by a radioactive or fluorescent label on the
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