BIOL 1411 1st Edition Lecture 29 Outline of Last Lecture I Mutations Outline of Current Lecture I Genetic Analysis II Relating Genotype with phenotype Lecture Costs and Benefits of Mutations o 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 load o 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 alleles o 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 Reaction o Copies of DNA sequences can be made by the polymerase chain reaction PCR technique o 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 template These 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 reaction o PCR generates many copies of the DNA fragment referred to as amplifying the sequence Copies of target region double each cycle o Complementary primers about 15 30 bases long are synthesized IDT according to known sequence Types of DNA sequence variation o 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 by side on chromosomes usually in noncoding regions Genetic Markers o 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 Electrophoresis o DNA fragments can be separated by sixe using gel electrophoresis o 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 end o 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 fragments o Mutations at restriction sites the change the ability of the enzyme to cut can be assayed as restriction fragment length polymorphisms o 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 allelespecific oligonucleotide hybridization method o Short synthetic DNA strands called oligonucleotide probes hybridize with denatured PCR products o Hybridization to the DNA containing the complementary SNP is detected by a radioactive or fluorescent label on the probe
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