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Ass. Lecturer : Khetam Qaid Mayee Genetic Microbiology practice / 3red Lecture / 5 Mutations A mutation is any physical change in the genetic material (such as a gene or a chromosome). A gene that contains a mutation ( change in the base sequence of the DNA) will produce an altered mRNA molecule that will produce an altered sequence of amino acids in the resulting protein General Types of Mutations Chromosomal Mutations 1. 2. Changes in chromosome structure Deletion, duplication, inversion, or translocation. Changes in chromosome number Polyploidy, aneuploidy (autosomes or sex chromosomes). a. Changes in the DNA of a gene made by the substitution of a single base with another or by addition or deletion of one or more nucleotides. Sickle cell disease results from a single base change Point Mutations Note : Remember! In RNA, the nucleotide base uracil replaces thymine. Point Mutations : Changes in single DNA nucleotides. o A missense mutation substitutes a different amino acid for the original one. Example: Sickle cell disease results from a single base change Remember! In RNA, the nucleotide base uracil replaces thymine. TEMPLATE DNA code CTC (Glutamine - glu) -mutation-> TEMPLATE DNA code CAC (valine - val) o A nonsense mutation results in a stop codon being inserted someplace before the end of the gene. TEMPLATE DNA code ATG (tyrosine - tyr) -mutation-> TEMPLATE DNA code ATT (STOP) o Silent mutations are point mutations that do not change the amino acid sequence of the protein. These are most likely to have no effect. Redundancy of the Genetic Code reduces the chance that point mutations that result in a change in the third nucleotide of a codon will alter the specified amino acid. The mRNA codons GAA and GAG code for the amino acid Glutamic Acid (Glu). The mRNA codons GCU, GCC, GCA, and GCG all code for the amino acid Alanine (Ala). The mRNA codons GGU, GGC, GGA, and GGG all code for the amino acid Glycine (Gly). o Frameshift Mutations: Additions or deletions of one or more nucleotides. Normal Sequence Mutation 1 CTG / TTA / CGC CTG / TTG / CGC Silent Mutation 2 CTG / TTT / CGC Missense Mutation 3 ATT / TTA / CGC Nonsense Template strand of the DNA to be transcribed Type of Mutation Mutation 4 CTA / GTT / ACG / C Addition (Frameshift) Mutation 5 CT_T / TAC / GC Deletion (Frameshift) Mutation 6 CTG / CTG / TTA / CGC Expansion Methods of detection mutations Screening Methods : In general, target sequences are amplified by PCR before analysis. At present, Taq polymerase is widely used for amplification. The error rate of Taq polymerase is in the range of 1024 to 1025 per nucleotide and is strongly affected by th e reaction conditions (e.g., concentrations of magnesium chloride and dNTPs, pH, and temperature). Depending on the method of choice, polymerase errors may contribute reasonably to unspecific background, limiting the level of detection. Denaturing gradient gel electrophoresis (dgge), temperature gradient . Double-stranded (ds) DNA is electrophoresed through a gradient of increasing concentration of a denaturing agent increasing concentration of denaturant or temperature, domains in the DNA dissociate according to their melting temperature (Tm). Chemical cleavage method (ccm). Enzyme mismatch cleavage (emc). RNase a cleavage method. Cleavase fragment length polymorphism (cflp). Protein truncation test (ptt).