Tilling Technique to fin d the mutation

2.  Name: Mohsin mukhtar
 REG NO: 2015-ag-7287
 DEGREE: BSc Hons Agrisciences (PBG)
 COURSE: MUTATION BREEDING
 TOPIC: TILLING
UAF-SUB Campus BUREWALA

3. TILLING
TARGETING INDUCED LOCAL LESIONS IN GENOME

4. Discovery of TILLING
TILLING first begin in the late 1990’s by McCallum
who work on the characterizing the function of the two
Chromomethylase gene in Arabidopsis.

5. Eco TILLING
DE-TILLING
 TILLING

6. WHAT IS TILLING?
• TILLING is general reverse genetics technique that
combines chemical mutagenesis with PCR based screening
to identify the chemically induced point mutations in region
of interest.
• TILLING is a powerful technology that employed
heteroduplex analysis to detect which organism in a
population carry single nucleotide mutation in specific gene.

7. DIFFERENCE
FORWARD GENETICS
• Phenotype
• Protein
• DNA sequence
REVERSE GENETICS
• DNA sequence
• Protein
• Phenotype

8.  Eco TILLING
Eco TILLING is similar to TILLING, that its objective is to
identify natural genetic variation as opposed to the induced mutation
 De-TILLING
A new knockout technique to obtain deletion mutants for target genes
a strategy to screen for rare deletion mutants in large fast neutron
mutagenised populations was first developed by Li et al. (2001)
and demonstrated in Arabidopsis and rice.

9. Heteroduplexes
A nucleic acid molecule (as DNA) composed of two chains with each
derived from a different parent molecule that are formed when
multiple alleles are amplified by PCR and are then heated and slowly
cooled. A “bubble” forms
at the mismatch of the two DNA strands, which is then cleaved by a
single stranded nucleases.

10. TILLING PROCEDURE
• There are different steps in TILLING Protocol.
1.Generation of the mutant population
2.Extraction of the DNA
3.Pooling
4.PCR or Amplification
5.Detection of heteroduplex
6.Heteroduplex Enzymatic cleavage
7.Phenotypic Recovery

11.  GENERATION OF MUTANT
POPULATION
IT IS 1ST AND MOST IMPORTANT REQUIREMENT
FOR THE TILLING.
TWO MAIN STEPS ARE TO BE CONSIDERED
BEFORE CHEMICAL APPLICATION.
1. CHEMICAL MUTAGEN THAT WORK BEST.
2. MATERIAL TO BE MUTAGENIZE.
EMS IS MOSTLY USED CHEMICAL MUTAGEN

12. GENERATION OF MUTANT
POPULATION
TILLING Purposes, The Utilisation Of Chemicals
With A Low Point-mutation Frequency Requires.
The Production Of Larger Populations To Allow For
A Sufficient Number Of Mutant Alleles Per Gene.

13. MUTAGENSIS

14. DNA EXTRACTION
Extraction of the DNA from the
mutagenize population M2
Then pool the DNA

17. DNA AMPLIFICATION BY USING PCR

18. DETECTION OF HETERODUPLEX
• Gene specific primers are available to find the sequence of gene of interest.
• Software are also use to identify the point mutation in a gene such as CODDLE
• Codons Optimized to Detect Deleterious LEsions
• Once a genomic sequence and the corresponding coding sequence is available,
CODDLE identifies regions where point mutations are most likely to cause deleterious
effects on gene function.
• Screening the mutant the population compare with wild type at a single nucleotide
level.
• Detection of the cleavage fragment was based on a Denaturing High Pressure
Liquid Chromatography (DHPLC).

19. HETERODUPLEX ENZYMATIC CLEAVAGE
• The products of amplification are then denatured and reannealed, generally by
heating and cooling. If a point mutation occurs in the amplified region in at least
one of the single DNA strands of the pool, it is likely that the mutant strand will
reanneal with the wild-type strand so that a fraction of the pool will have a
mismatch at the site of the mutation. And the find the mismatch in heteroduplex.
• After the amplification of the desired sequence of heteroduplex we cleavage or
cut the at point of mutation with the enzyme of S1 nuclease family Cel1.

21. PHENOTYPE RECOVERY
• When allelic series of mutation discovered through
TILLING
• Phenotypic analysis is required to verify the effect of
mutation.

22. MERITS AND DEMERITS OF TILLING
MERITS
• Cheap and rapid reverse genetic
approach
• Firstly use to detect a chemical
mutagenesis at single nucleotide level
• It is independent of genome size,
reproductive system or generation
time.
• Valuable for essential gene
DEMERITS
• Skilled labour is required
• Depend upon primary design
• Lower rate of induction of mutation

23. Contents taken from that book