RECOMBINANT DNA TECHNOLOGY

PROJECT BASED LEARNING

TOPIC- MUTAGENESIS

VIKAS SHARMA

20915002

Mutagenesis is the formation of mutations where the genetic information has been changed of an
organism. Mutagenesis occurs spontaneously in nature, resulting change in DNA which consists of
nucleotides that contain a phosphate backbone, a deoxyribose sugar, and one of four nitrogen-containing
bases (adenine [A], guanine [G], cytosine [C], and thymine [T]). Mutagenesis is the driving force of
evolution; however, it can also lead to cancers and heritable diseases. 1

Sickle cell disease is the most common example of single base-pair substitutions mutation where single
base –pair mutation results in glutamate to valine amino acid substitution.

Mutagenesis is the driving force behind evolution, and genetic variation is the result of mutations. Any
change in genetic information may result in advantageous or disadvantageous phenotypic characteristics
that impact an organism's fitness. When a mutation results in a higher fitness, natural selection favors
these phenotypes, and these traits are more likely to be passed to offspring. 2

Site-directed Mutagenesis
SDM is a technique where DNA can be modified at a specific nucleotide location, causing a
predetermined amino acid change. These substitution mutations can result in drastic changes in protein
conformation and function. This technique requires

(1) A DNA template with a target gene,

(2) Knowledge of the target gene's nucleotide sequence,
(3) A short primer (commonly 20 to 30 base pairs) complementary to the target sequence that is
modified to contain a mismatched nucleotide (typically 1 to 3 base pairs that will cause an amino
acid change).

1
Zhang L, Vijg J. Somatic Mutagenesis in Mammals and Its Implications for Human Disease and Aging. Annu Rev
Genet. 2018 Nov 23;52:397-419.
2
Lenski RE. What is adaptation by natural selection? Perspectives of an experimental microbiologist. PLoS
Genet. 2017 Apr;13(4):e1006668.
 Step 1: Separation of the two strands of template DNA. This can be accomplished by heat or an
alkali solution.

Step 2: Addition of the modified DNA primer. Once the DNA primer anneals with a single
strand, a DNA polymerase replicates the strand.

Step 3: The second round of replication yields a mutant DNA strand that can be used to
synthesize a modified protein.

Application
Crop Improvement

Mutagenesis is one of the most efficient tools that has been utilized extensively to create genetic variation
as well as for identification of key regulatory genes for economically important traits toward the crop
improvement. It is a promising approach to develop new varieties with improved agronomic
characteristics, such as higher stress tolerance potential (biotic and abiotic stress) and bio-fortification. 3

The general strategy used to create a mutant population of self-fertilizing crops (e.g. barley, durum,
wheat, and rice) as a resource for both forward and reverse genetic approaches is indicated. Most
mutagenized populations are generated by exposing seeds (M 0) to the mutagen and allowing the resultant
M1 plants produced to self-fertilize and give rise to M 2 seed. The seeds must be exposed to sufficient
mutagen to ensure a high level of mutations but without affecting viability and fertility. To ensure the
greatest number of unique novel mutations it is recommended that only one seed is taken from each
M1 plant. Leaf material is taken from the resultant M 2 plants for the isolation of genomic DNA that is
used as the resource for mutation detection. The M 2 plants are allowed to mature and the M3 seeds are

3
Mutagenesis as a tool in plant genetics, functional genomics, and breeding.Sikora P, Chawade A,
Larsson M, Olsson J, Olsson O Int J Plant Genomics. 2011; 2011():314829.
archived so that gene function can be studied in any plants in which mutations are identified. However, at
this stage, the population is still segregating and not all M 3 plants will carry the mutations identified in the
M2 parent. The mutagenized population may be taken through further generations by single seed descent
to generate near-homozygous material (∼3% heterozygous mutations at M6), although up to half of all
mutations present in the M1 are lost in the process. The mutagenized populations also form a valuable
resource for forward genetic screening approaches for traits such as plant architecture, yield, quality,
resource use efficiency, stress tolerance, and pest and pathogen resistance. Detailed and comprehensive
documentation of phenotypes is essential in order to increase the opportunity for the incorporation of
novel traits into breeding programmes 

Mutant lines, whether identified by forward or reverse screening, will also be carrying numerous
additional mutations in addition to that selected. For a diploid species such as rice with its relatively small
genome the number of extraneous mutation may well be in the tens of thousands, and in the case of a
mutation-tolerant species such as bread wheat, it is estimated that in any one M 2 individual within our
population the total number of mutations may approach half a million. While the vast majority of these
probably have no effect, it is obviously unwise to conclude that any observed phenotype observed at
M3 or later generations is due to the single point mutation under study. At the very least, it is essential to
show genetic linkage of the mutation and phenotype, and some degree of back-crossing will almost
certainly be necessary.4

4
https://academic.oup.com/jxb/article/60/10/2817/579244