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“T-DNA & Transposon Tagging”
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 INTRODUCTION
 ROLE OF GENE TAGGING
 TRANSPOSON TAGGING
 TRANSPOSON TAG...
• Gene tagging strategies are used to isolate those genes that
produce a detectable phenotype.
• Gene tagging broadly invo...
• Gene tagging is the method of insertion a recognizable DNA
fragment with in a gene which a gene such that the function o...
• These DNA inserts are then analyzed to identify and isolate the
gene responsible for the mutant phenotype produced by
in...
“T-DNA & Transposon Tagging”
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Flow chart 1 - A generalized scheme for gene tagging
• Transposon tagging describes isolation of genes using
transposable elements as gene tags.
• This strategy is applicable ...
“T-DNA & Transposon Tagging”
• When a transposon integrates within a gene, the gene function is lost.
• But when the trans...
“T-DNA & Transposon Tagging”
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Fig. 2- Transposons tagging
• It is necessary to establish that the mutational event is due to transposon
insertion; in this respect, the instability ...
• Agrobacterium T-DNA integrates into
plant genome at random sites.
• Agrobacterium mediated transformation
can be used to...
• Transposon tagging has been used to isolate several genes in maize (e.g.
A1, A2, BZ2, C1, C2, opaque2, R, P, etc.), toma...
• Gene tagging strategies are used to isolate those genes that produce a
detectable phenotype, but whose base sequences or...
• T-DNA and transposon tagging are very important technique
for isolation of unknown DNA sequences.
• These strategies are...
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Primrose S. 2004 Principle Of Gene Manupulation
6th Edition
Lewin Benjamin 2007 Gene IX
9th Edition
“T-DNA & Transposon...
THANK
YOU
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T dna & transposone tagging 1 (2)

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T dna & transposone tagging 1 (2)

  1. 1. 1
  2. 2. 2 “T-DNA & Transposon Tagging” S Y N O P S I S  INTRODUCTION  ROLE OF GENE TAGGING  TRANSPOSON TAGGING  TRANSPOSON TAGGING OF PLANT GENES  DIFFICULTIES IN TRANSPOSON TAGGING  T-DNA TAGGING  SUMMARY  CONCLUSION  REFERENCES
  3. 3. • Gene tagging strategies are used to isolate those genes that produce a detectable phenotype. • Gene tagging broadly involve the insertion of a recognizable DNA fragment with a gene. • T-DNA is the part of ti-plasmid , DNA found in the soil bacterium. • Transposons are mobile genetic element that can move from one place to another place in a DNA molecule. • T-DNA and transposons can be used in gene tagging and gene analysis. “T-DNA & Transposon Tagging” I N T R O D U C T I O N 3
  4. 4. • Gene tagging is the method of insertion a recognizable DNA fragment with in a gene which a gene such that the function of the gene is distrupted. • Gene can be easily recognize by virtue of the inserted fragment. • The inserted fragment is usually a well characterized transposable element, most of which has been sequenced. “T-DNA & Transposon Tagging” R O L E O F G E N E T A G G I N G 4
  5. 5. • These DNA inserts are then analyzed to identify and isolate the gene responsible for the mutant phenotype produced by insertional mutagenesis. • The tag may be based on – T-DNA of Agrobacterium – A transposable element – A retroviral genome “T-DNA & Transposon Tagging” R O L E O F G E N E T A G G I N G 5
  6. 6. “T-DNA & Transposon Tagging” R O L E O F G E N E T A G G I N G 6 Flow chart 1 - A generalized scheme for gene tagging
  7. 7. • Transposon tagging describes isolation of genes using transposable elements as gene tags. • This strategy is applicable to both plants as well as animals, but we shall confine on discussion to their use in plants. • A transposable element is a DNA sequence that has the ability to change its location in the genome, i.e., it can transpose from one location to another in the genome. • Some transposable elements behave like retroviruses and, for this region, they are called retrotransposons. “T-DNA & Transposon Tagging” T R A N S P O S O N E T A G G I N G 7
  8. 8. “T-DNA & Transposon Tagging” • When a transposon integrates within a gene, the gene function is lost. • But when the transposon move out of the gene, the gene function may be partially of fully restored. • Thus when a Ds element integrates within a gene, the gene function may be partially or fully restored. • Thus when a Ds element integrates within the gene C1 on chromosome 9 of maize, C1 function is suppressed and colourless kernels are produced. • Several maize inbred lines having colourless kernels are of this type. • The Ds may also transpose in the germline; in such a case, the functional C1 gene will be transmitted to the next generation. 8 T R A N S P O S O N E T A G G I N G O F P L A N T G E N E S
  9. 9. “T-DNA & Transposon Tagging” 9 T R A N S P O S O N E T A G G I N G O F P L A N T G E N E S Fig. 2- Transposons tagging
  10. 10. • It is necessary to establish that the mutational event is due to transposon insertion; in this respect, the instability of mutant phenotype may serve as a useful marker. • Species like maize and snapdragons carry several different transposon, and each transposon, and may be parent in more than one copy per genome. • A major limitation of the method is the low frequency of transposition. • In addition, most species lack active transposons. “T-DNA & Transposon Tagging” D I F F I C U L T I E S 10
  11. 11. • Agrobacterium T-DNA integrates into plant genome at random sites. • Agrobacterium mediated transformation can be used to produce a large number of independent transformants. • Insertion of T-DNA within a gene would generates a mutant phenotype. • The transformants are screened for mutant phenotype, and the genomic DNAs of the selected mutants can then be used for isolation of the concerned gene by using the T-DNA sequence either as a probe or as primers. “T-DNA & Transposon Tagging” T- D N A T A G G I N G 11 Fig. 3 - Ti plasmid
  12. 12. • Transposon tagging has been used to isolate several genes in maize (e.g. A1, A2, BZ2, C1, C2, opaque2, R, P, etc.), tomato (cf-9, Dem, etc.), tobacco (cf-4A), rice (Ei-Ef-1), etc. • In gene expresion. • In gene silencing. • In knockout gene. • In site directed mutagenesis. “T-DNA & Transposon Tagging” A P P L I C A T I O N S 12
  13. 13. • Gene tagging strategies are used to isolate those genes that produce a detectable phenotype, but whose base sequences or protein products are not known. • The tag may be based on – T-DNA of Agrobacterium – A transposable element – A retroviral genome • Transposon tagging has been used to isolate several genes in maize (e.g. A1, A2, BZ2, C1, C2, opaque2, R, P, etc.), tomato (cf-9, Dem, etc.), tobacco (cf-4A), rice (Ei-Ef-1), etc. “T-DNA & Transposon Tagging” S U M M A R Y 13
  14. 14. • T-DNA and transposon tagging are very important technique for isolation of unknown DNA sequences. • These strategies are used for the construction of genomic libraries. 14 “T-DNA & Transposon Tagging” C O N C L U S I O N
  15. 15. 15 Primrose S. 2004 Principle Of Gene Manupulation 6th Edition Lewin Benjamin 2007 Gene IX 9th Edition “T-DNA & Transposon Tagging” R E F E R E N C E S
  16. 16. THANK YOU

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