PCR screen

Detecting sequence specific Mu insertions by PCR
By analysing plants with Mu insertions within specific sequences, we may gain a better understanding of the role of these sequences and proteins which they may encode. We have designed a PCR based screen to identify plants with Mu insertions up to 1000 bp from a known sequence.

Primary Screen
The primary screen identifies which of the 100 pools of 50 plants contain a specific Mutator insertion event. Each of the pools of genomic DNA is PCR screened using a gene specific and a Mutator specific primer. Products are dot blotted onto nylon membrane and detected by hybridisation with a gene specific probe. Duplicate PCR is required due to the high incidence of false positives. Repeated primary screens using nested, gene specific primers allows confirmation of the insertion event. Primary screens frequently indicate between one and five pools which may then undergo a secondary screen.

Secondary Screen
The secondary screen identifies which of the 50 plants within primary screen pool contains the Mutator insertion. DNA is extracted from pairs of plants producing 25 genomic pools. PCR of these samples using the primers from the primary screen, followed by agarose gel electophoresis, narrows the insertion event to one of two plants. The size of the PCR product corresponds to distance between the insertion site and the gene specific primer while sequencing the product confirms the exact insertion site. Kernels from both secondary screen positives are planted and seedlings re-screened by PCR to identify progeny containing the insertion.

Graphical overview

Primary screen identifies which DNA pools contain specific insertion events.

Secondary screen identifies the individual plant containing the mutator insert and its distance from the gene primer.

The product from the secondary screen is sequenced to confirm the exact insertion point.

Seed from the positive individual is backcrossed and the phenotype assessed.

Back to top

Supported by:

Maintained by gary.barker@bristol.ac.uk Last updated Dec 2001