(自学)体外定点突变
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体外定点突变
Why?
Want to determine how DNA and/or encoded proteins
function in intact entity (virus, bacterium, cell, animal etc.)
Most direct way to find out what a gene or protein does is
Types of Mutation (2)
LETHAL MUTATION: causes developing organism to die
prematurely
CONDITIONAL MUTATION: produces its phenotypic
effect only under certain conditions, called the restrictive conditions. Under other conditions—the permissive conditions—the effect is not seen. For a temperature-sensitive mutation, the restrictive condition typically is high temperature, while the permissive condition is low temperature abolishes the activity of the gene. This is the most common class of mutation. Loss-of-function mutations are usually recessive—the organism can usually function normally as long as it retains at least one normal copy of the affected gene
mutagenic oligo By definition, oligo sequence incorporates at least one base change May be far more complicated including insertions, deletions and compound substitutions Minimum length of oligo determined by complexity of mutation Simple single base mutations use ~25 nt oligos More complicated mutations may require oligos of >80 nts Efficiency of mutagenesis optimized by design of; Base sequence Base composition Melting temperature Propensity to form secondary structures Specificity of annealing
LOSS-OF-FUNCTION MUTATION: either reduces or
Types of Mutation (3)
NULL MUTATION: loss-of-function mutation completely
abolishing activity of gene
GAIN-OF-FUNCTION MUTATION: increases activity of
DOMINANT NEGATIVE MUTATION: dominant-acting
SUPPRESSOR MUTATION: suppresses phenotypic
effect of another mutation, so double mutant seems normal:
In Vitro Mutagenesis(体外基因突变)
How?
GENETIC SCREEN… Select PHENOTYPE – determine GENOTYPE or
REVERSE GENETICS… Create GENOTYPE – determine PHENOTYPE i.e., translate sequence into function
INVERSION MUTATION: inverts segment of
DELETION: deletes segment of chromosome TRANSLOCATION: breaks off segment from one
chromosome and attaches it to another
Classical Site-Directed Mutagenesis
Protocols for site-directed mutagenesis involve; Design and synthesis of mutagenic
oligonucleotides Hybridization of mutagenic oligo to ssDNA target cloned into bacteriophage vector (usually M13) – eliminates competition between mutagenic oligo and complementary DNA strand Extension of hybridized oligo by DNA polymerase with all 4 dNTPs Formation of closed circular DNA by ligation with DNA ligase Transfection of susceptible bacteria Screening for mutant clones by hybridization (e.g., using oligo) – sequence confirmation Recovery of mutated DNA fragment Substitution of mutagenized fragment for corresponding wt DNA sequence
At its most simplistic, in vitro mutagenesis allows us
to change the base sequence of a DNA segment or gene Mutations can be localized or general, random or targeted; Less specific methods of mutagenesis used to analyze regulatory regions of genes More specific methods of mutagenesis used to understand contribution of individual amino acids, or groups of amino acids, to structure and function of target protein Both methods generate mutants in vitro, without phenotypic selection
to find out what happens when it is missing or mutated
Study mutants that lack gene/protein or express altered
version of it - determine which biological processes are altered in mutants Change solubility, stability, structure, function of a protein Change enzyme activity or substrate specificity
gene or makes it active in inappropriate circumstances; usually dominant mutation blocking gene activity, causes loss-of-function phenotype even in presence of normal copy of gene. Occurs when mutant gene product interferes with function of normal gene product
Traditional general procedure
Generate ssDNA (M13) Anneal mutagenic oligo Extend with DNA polymerase
and dNTP Seal nicks with DNA ligase Select for mutant strand or deseBaidu Nhomakorabeaect for wild type strand Transform and screen Isolate DNA and sequence to verify
体外基因突变(In Vitro Mutagenesis) 包括：
单个碱基或片断的替换 基因片断的插入 基因片断的删除
根据其特点可将体外基因突变技术分为两大类：
位点特异性突变 随机突变
定点突变
位点特异性突变的类型
寡核苷酸介导的基因突变指用含有突变碱基的
寡聚核苷酸片断作为引物，在聚合酶的作用下 启动DNA分子进行复制。
1. Clone insert into plasmid vector
2. Denature and anneal mutagenic oligonucleotides
3. Extend using DNA polymerase; Ligate using T4 DNA ligase
4. Select mutant strand; Retransform into final host
Classical Site-Directed Mutagenesis
Proportion of clones containing mutation 0.1% -
50% depending on efficiency of mutagenic oligo Low efficiency Several methods developed to select against clones containing non-mutant DNA including; Selective digestion of template DNAs by removal of modified bases (KUNKEL MUTAGENESIS), Elimination of restriction site from mutant DNAs (USE MUTAGENESIS) Selective digestion of template DNAs by protection of modified bases (Phosphorothiate method ), using restriction enzymes or exonucleases
Types of Mutation (1)
POINT MUTATION: maps to single site in genome,
corresponding to single nucleotide pair or very small part of single gene
chromosome
盒式突变是利用一段人工合成的含基因突变序
列的寡核苷酸片段，取代野生型基因中的相应 序列。
PCR介导的基因突变
Design of Mutagenic Oligonucleotides
Crucial step in site-directed mutagenesis is design of
Why?
Want to determine how DNA and/or encoded proteins
function in intact entity (virus, bacterium, cell, animal etc.)
Most direct way to find out what a gene or protein does is
Types of Mutation (2)
LETHAL MUTATION: causes developing organism to die
prematurely
CONDITIONAL MUTATION: produces its phenotypic
effect only under certain conditions, called the restrictive conditions. Under other conditions—the permissive conditions—the effect is not seen. For a temperature-sensitive mutation, the restrictive condition typically is high temperature, while the permissive condition is low temperature abolishes the activity of the gene. This is the most common class of mutation. Loss-of-function mutations are usually recessive—the organism can usually function normally as long as it retains at least one normal copy of the affected gene
mutagenic oligo By definition, oligo sequence incorporates at least one base change May be far more complicated including insertions, deletions and compound substitutions Minimum length of oligo determined by complexity of mutation Simple single base mutations use ~25 nt oligos More complicated mutations may require oligos of >80 nts Efficiency of mutagenesis optimized by design of; Base sequence Base composition Melting temperature Propensity to form secondary structures Specificity of annealing
LOSS-OF-FUNCTION MUTATION: either reduces or
Types of Mutation (3)
NULL MUTATION: loss-of-function mutation completely
abolishing activity of gene
GAIN-OF-FUNCTION MUTATION: increases activity of
DOMINANT NEGATIVE MUTATION: dominant-acting
SUPPRESSOR MUTATION: suppresses phenotypic
effect of another mutation, so double mutant seems normal:
In Vitro Mutagenesis(体外基因突变)
How?
GENETIC SCREEN… Select PHENOTYPE – determine GENOTYPE or
REVERSE GENETICS… Create GENOTYPE – determine PHENOTYPE i.e., translate sequence into function
INVERSION MUTATION: inverts segment of
DELETION: deletes segment of chromosome TRANSLOCATION: breaks off segment from one
chromosome and attaches it to another
Classical Site-Directed Mutagenesis
Protocols for site-directed mutagenesis involve; Design and synthesis of mutagenic
oligonucleotides Hybridization of mutagenic oligo to ssDNA target cloned into bacteriophage vector (usually M13) – eliminates competition between mutagenic oligo and complementary DNA strand Extension of hybridized oligo by DNA polymerase with all 4 dNTPs Formation of closed circular DNA by ligation with DNA ligase Transfection of susceptible bacteria Screening for mutant clones by hybridization (e.g., using oligo) – sequence confirmation Recovery of mutated DNA fragment Substitution of mutagenized fragment for corresponding wt DNA sequence
At its most simplistic, in vitro mutagenesis allows us
to change the base sequence of a DNA segment or gene Mutations can be localized or general, random or targeted; Less specific methods of mutagenesis used to analyze regulatory regions of genes More specific methods of mutagenesis used to understand contribution of individual amino acids, or groups of amino acids, to structure and function of target protein Both methods generate mutants in vitro, without phenotypic selection
to find out what happens when it is missing or mutated
Study mutants that lack gene/protein or express altered
version of it - determine which biological processes are altered in mutants Change solubility, stability, structure, function of a protein Change enzyme activity or substrate specificity
gene or makes it active in inappropriate circumstances; usually dominant mutation blocking gene activity, causes loss-of-function phenotype even in presence of normal copy of gene. Occurs when mutant gene product interferes with function of normal gene product
Traditional general procedure
Generate ssDNA (M13) Anneal mutagenic oligo Extend with DNA polymerase
and dNTP Seal nicks with DNA ligase Select for mutant strand or deseBaidu Nhomakorabeaect for wild type strand Transform and screen Isolate DNA and sequence to verify
体外基因突变(In Vitro Mutagenesis) 包括：
单个碱基或片断的替换 基因片断的插入 基因片断的删除
根据其特点可将体外基因突变技术分为两大类：
位点特异性突变 随机突变
定点突变
位点特异性突变的类型
寡核苷酸介导的基因突变指用含有突变碱基的
寡聚核苷酸片断作为引物，在聚合酶的作用下 启动DNA分子进行复制。
1. Clone insert into plasmid vector
2. Denature and anneal mutagenic oligonucleotides
3. Extend using DNA polymerase; Ligate using T4 DNA ligase
4. Select mutant strand; Retransform into final host
Classical Site-Directed Mutagenesis
Proportion of clones containing mutation 0.1% -
50% depending on efficiency of mutagenic oligo Low efficiency Several methods developed to select against clones containing non-mutant DNA including; Selective digestion of template DNAs by removal of modified bases (KUNKEL MUTAGENESIS), Elimination of restriction site from mutant DNAs (USE MUTAGENESIS) Selective digestion of template DNAs by protection of modified bases (Phosphorothiate method ), using restriction enzymes or exonucleases
Types of Mutation (1)
POINT MUTATION: maps to single site in genome,
corresponding to single nucleotide pair or very small part of single gene
chromosome
盒式突变是利用一段人工合成的含基因突变序
列的寡核苷酸片段，取代野生型基因中的相应 序列。
PCR介导的基因突变
Design of Mutagenic Oligonucleotides
Crucial step in site-directed mutagenesis is design of