Mutations Lecture Notes PDF

Muta%ons: Primary subject and tool of gene%c analysis Muta%ons are heritable changes in base sequences that modify the informa%on content of DNA. – Natural gene%c varia%on – Induced muta%ons Random mutagen Specific target  Classifica%on of muta%ons by affect on DNA mo...

Muta%ons: Primary subject and tool of gene%c analysis Muta%ons are heritable changes in base sequences that modify the informa%on content of DNA. – Natural gene%c varia%on – Induced muta%ons Random mutagen Specific target  Classifica%on of muta%ons by affect on DNA molecule Starting sequence Subs%tu%on – base is T C T C G C A T G G T A G G T replaced by one of the other A G A G C G T A C C A T C C A three bases Type of mutation and effect on base sequence Dele%on – block of one or (a) Substitution more DNA pairs is lost Transition: Purine for purine, pyrimidine for pyrimidine T C T C G C A T A G T A G G T Inser%on – block of one or A G A G C G T A T C A T C C A Transversion: Purine for pyrimidine, pyrimidine for purine more DNA pairs is added T C A C G C A T G G T A G G T A G T G C G T A C C A T C C A Inversion – 180o rota%on of piece of DNA (b) Deletion T C T C T G G T A G G T Reciprocal transloca%on – A G A G A C C A T C C A G C A parts of nonhomologous C G T chromosomes change places (c) Insertion A A Chromosomal T T rearrangements – affect T C T C A A G C A T G G T A G G T A G A G T T C G T A C C A T C C A many genes at one %me7-3 (d) Inversion Site of inversion 5' 3' (b) Deletion T C T C T G G T A G G T A G A G A C C A T C C A G C A C G T Classifica%on of muta%ons by affect on DNA (c) Insertion A A T T molecule T C T C A A G C A T G G T A G G T A G A G T T C G T A C C A T C C A Subs%tu%on – base is (d) Inversion Site of inversion replaced by one of the other 5' T C T C G C A T G G T A G G T A G A G C G T A C C A T C C A 3' three bases 3' 5' 3' Dele%on – block of one or 5' T A C C A T G C G A T G G T A C G C more DNA pairs is lost 5' 5' 3' 3' Inser%on – block of one or 3' T C T T A C C A T G C G G G T A G A A T G G T A C G C C C A 5' more DNA pairs is added Inversion – 180o rota%on of (e) Reciprocal translocation Chromosome 1 Chromosome 2 piece of DNA Reciprocal transloca%on – T TA A GGC T A A T T C CG A Chromosome A T C G C TA A T A G C GA TT parts of nonhomologous breaks chromosomes change places Translocation – Chromosomal rearrangements – affect many T T A A C TA A A TC G GGC T genes at one %me A A T T GA T T TA G C C CGA 7-4  Spontaneous muta%ons influencing phenotype occur at a very low rate. Muta%on rates from wild‐type to recessive alleles for five coat color genes in mice Natural processes change informa%on stored in DNA Hydrolysis of a purine base, Deamina%on removes –NH2 A or G occurs 1000 %mes group. Can change C to U, an hour in every cell. inducing a subs%tu%on to an A‐T base pair aTer replica%on  X‐rays break the DNA backbone. UV light produces thymine dimers. Oxida%on from free radicals formed by irradia%on damages individual bases. Mistakes during replica%on alter gene%c informa%on Errors during replica%on are exceedingly rare, less than once in 109 base pairs. Proofreading enzymes correct errors made during replica%on. – DNA polymerase has 3’ – 5’ exonuclease ac%vity which recognizes mismatched bases and excises it. Unequal crossing over creates one homologous chromosome with a duplica%on and the other with a dele%on Transposable elements move around the genome and are not suscep%ble to excision or mismatch repair. Mutagens increase muta%on rate using different mechanisms. Repair enzymes fix errors created by muta%on. Excision repair enzymes release damaged regions of DNA. Repair is then completed by DNA polymerase and DNA ligase. The Direct Detec%on of Genotype Dis%nguishes Individual Genomes Members of the same species show enormous sequence varia%on in their genomes. 1 in 1000 bp differ in any two randomly chosen humans. Since the haploid genome is 3 × 109 bp, 3 million differences between any two randomly chosen individuals Most differences are in non‐coding, nonregulatory regions. Alleles – any varia%ons in the genome at a par%cular loca%on (locus) Polymorphic – two or more alleles at a locus Polymorphism – the par%cular varia%on DNA marker – polymorphic locus useful for mapping studies, disease diagnosis Anonymous locus – posi%on on genome with no known func%on Four classes of DNA polymorphisms Single nucleo%de polymorphism (SNP) Single base‐pair subs%tu%ons Arise by mutagenic chemicals or mistakes in replica%on Biallelic – only two alleles Ra%o of alleles ranges from 1:100 to 50:50. 2001 – over 5 million human SNPs iden%fied Most occur at anonymous loci. Muta%on rate of 1 X 10‐9 per locus per genera%on Very few are thus new muta%on in the species. Useful as DNA markers  Microsatellites 1 every 30,000 bp (varies) Repeated units 2 – 5 bp in length Mutate by replica%on error Muta%on rate of 10‐3 per locus per gamete Useful as highly polymorphic DNA markers Polymerase chain reac%on to rapidly isolate and amplify DNA fragments PCR (polymerase chain reac%on) achieved exponen%al accumula%on of target DNA – Based on previously determined DNA sequence, develop short oligonucleo%des (~ 20bp) complementary to sequences flanking the target DNA. – Oligonucleo%des act as primers to copy DNA similar to DNA replica%on. Oligonucleo%de primers begin copying DNA Each cycle of replica%on doubles amount of target DNA Exponen%al amplifica%on Microsatellite allele detec%on analysis of size differences Gel electrophoresis separates DNA fragments according to size. Preparing an agarose gel for electrophoresis Gel electrophoresis separates DNA fragments according to size. Loading DNA fragments onto an agarose gel and performing electrophoresis Gel electrophoresis separates DNA fragments according to size. Visualizing DNA in an agarose gel Dele%ons, duplica%ons, and inser%ons Expand or contract the length of nonrepe%%ve DNA Small dele%ons and duplica%ons arise by unequal crossing over Small inser%ons can also be caused by transposable elements Much less common than other polymorphisms Fragmen%ng complex genomes into pieces for analysis Restric%on enzymes fragment the genome at specific sites.  SNP detec%on by PCR Must have sequence on either side of polymorphism – Amplify fragment – Expose to restric%on enzyme – Gel electrophoresis e.g., sickle‐cell genotyping with a PCR based protocol