This document discusses forward and reverse genetic approaches for understanding gene function. Forward genetics begins with a phenotype and identifies the underlying gene, while reverse genetics starts with a gene and determines its phenotype. Specific reverse genetic techniques described include large-scale random mutagenesis, homologous recombination, transposable element excision, RNA interference, genome editing using ZFNs/TALENs/CRISPR, and site-directed mutagenesis combined with transgenics. The document provides details on how each technique is used to alter genes and study their function.

1. Forward and Reverse genetic
approaches
MBB:601 Advances in plant molecular biology 3+0
1
Presented by
Ekatpure Sachin Chandrakant
PhD research Scholar
Department of Plant Biotechnology

2. 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
2

3. 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
3

4. 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
4

5. 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
5

6. 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
6

7. 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
7

8. 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
8

9. 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
9

10. Homologous recombination
10

11. 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.
11

12. 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”
12

13. RNA interference
13

14. 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
14

15. 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
15

16. 16