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Forward and Reverse genetic approaches

MBB:601 Advances in plant molecular biology 3+0

Presented byEkatpure Sachin Chandrakant

PhD research ScholarDepartment of Plant Biotechnology

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Introduction

• In molecular biology there are number of techniques are available to understand the function of the gene

• For identification of gene function there are two methods used commonly

– Forward genetics (Classical genetics)

– Reverse genetics

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1. Forward genetics

• A traditional approach to the study of gene function that begins with a phenotype (a mutant organism) and proceeds to a gene that encodes the phenotype

• It depends upon the identification and isolation of random mutation that affect the phenotype of interest

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Forward genetics cont…

• Initially scientist are depends on the mutation that are occurs naturally, but after the discovery of mutagenic agent increases the rate of mutation

• First experimentally created mutation - using X rays - to induce X linked mutation in Drosophila melanogaster- by H. J. Muller in 1927

• Two types of mutations were majorly used in the forward mutation

– A. Spontaneous mutation

– B. Creating random mutation

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A. Spontaneous Mutation

• It arises spontaneously from natural changes in DNA structure or from error in the replication

• i.e. mutation results from both internal and external factors

• Where as the changes caused by the radiation or environmental chemicals are called as induced mutation

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B. Creating random mutation

• It depends upon the identification and isolation of random mutation that affect the phenotype

• Radiation (X rays), chemical mutagen (EMS) and transposable elements (insert within a coding region and disrupt the amino acid sequence ) are used to create the mutation

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2. Reverse genetics

• A molecular approach that begins with a genotype ( a DNA sequence) and proceeds to the phenotype by altering the sequence or by inhibiting its expression

• It is possible due to the advancement in the molecular genetics

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a) Large-scale random mutagenesis and screening

• Use forward mutagenesis (e.g. EMS), except instead of screening for a particular phenotype, screen your gene of interest for nucleotide changes

• Typically requires that screen 1000’s or 10,000’s of individuals

• This is done by performing PCR for gene of interest and looking for slight differences in the migration of the PCR product on a gel or column

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b. homologous recombination

• Recombination is the exchange of genetic information between DNA molecules; when the exchange is between homologous DNA molecules it is called homologous recombination

• Works in bacteria, yeast, mice and other mammals

• This method has been used to knockout every predicted ORF in yeast

• Many mouse genes have been knocked out by this method

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Homologous recombination

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c. Transposable element excision

• When a source of transposase is introduced, the TE will excise with some frequency resulting in a loss of the marker gene

• Often the TE excision will also result in a deletion of the flanking DNA.

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d. RNA interference (RNAi)• RNA interference (RNAi) is a biological process in which RNA molecules inhibit

gene expression, typically by causing the destruction of specific mRNA molecules

• Previously, it was known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling

• Double stranded RNA (dsRNA) can lead to specific post transcriptional gene silencing (PTGS)

• This mechanism is part of a natural response of the host that most likely evolved to control virus or TE replication

• Sometimes RNAi does not completely eliminate expression of the target gene, but only reduces it

• In these cases, it is referred to as a “knock down” instead of a “knock out”

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RNA interference

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e. Genome editing• Several methods have been developed to target mutations to a

specific location in the genome

• These can be used to knock-out target genes, make specific point mutations in a target gene, or even insert new genes or DNA sequences at a specific target site in the genome

• E.g.

– Zinc-finger nucleases (ZFNs)

– Transcription activator-like effector nucleases (TALENs)

– Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) nucleases

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f. Site-directed mutagenesis and transgenics

• Can make a specific nucleotide changes at an exact site in gene of interest or in DNA

• Requires: Gene of interest cloned into plasmid, host with null background (knockout)

• Mutagenesis is performed in vitro on the cloned gene in a plasmid replicated in bacteria

• Then the mutated gene is inserted into the host genome in a

transposable element vector

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