SRAP MARKER

Sequence-related amplified polymorphism (SRAP) : 

Sequence-related amplified polymorphism (SRAP) A.K. Chhabra

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Sequence-related amplified polymorphism (SRAP) G. Li · C. F. Quiros Theor Appl Genet (2001) 103:455–461 Developed in 2000 SRAP is aimed for the amplification of open reading frames (ORFs). It is based on two-primer amplification. The primers are 17 or 18 nucleotides long and consist of the following elements: Core sequences, which are 13 to 14 bases long, where the first 10 or 11 bases starting at the 5′ end, are sequences of no specific constitution (“filler” sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. The core is followed by three selective nucleotides at the 3′ end. The filler sequences of the forward and reverse primers must be different from each other and can be 10 or 11 bases long. Example of forward primers: me1, 5′-TGAGTCCAAACCGGATA-3′, me2, 5′-TGAGTCCAAACCGGAGC-3′, me3, 5′-TGAGTCCAAACCGGAAT-3′, me4, 5′-TGAGTCCAAACCGGACC-3′, me5, 5′-TGAGTCCAAACCGGAAG-3′. Example of reverse primers: em1, 5′-GACTGCGTACGAATTAAT-3′, em2, 5′-GACTGCGTACGAATTTGC-3′, em3, 5′-GACTGCGTACGAATTGAC-3′, em4, 5′-GACTGCGTACGAATTTGA-3′, em5, 5′-GACTGCGTACGAATTAAC-3′, em6, 5′-GACTGCGTACGAATTGCA-3′.

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Sequence-related amplified polymorphism (SRAP) The only rules for construction of the forward and reverse primers are that they do not form hairpins or other secondary structures, and to have a GC content of 40–50%. Exons are normally in GC-rich regions. Rules for construction of the forward and reverse primers:

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Sequence-related amplified polymorphism (SRAP) The purpose for using the “CCGG” sequence in the core of the first set of SRAP primers was to target exons to open reading frame (ORF) regions. This rationale is based on the fact that exons are normally in GC-rich regions. Exons are normally in GC-rich regions. Why CCGG is used in forward primer:

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Sequence-related amplified polymorphism (SRAP) Exons are normally in GC-rich regions. For example, in completed sequences of the chromosomes 2 and 4 of Arabidopsis thaliana (L.) Heynh., the GC content of exons is 43.6% and 44.08%, respectively. In introns, these values drop to 32.1% and 33.08%, respectively (Lin et al. 1999; The EU Arabidopsis Genome 1999). Examples of GC Rich Exons :

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Sequence-related amplified polymorphism (SRAP) Exons are normally in GC-rich regions. However, since exonic sequences are generally conserved among different individuals (Quiros et al. 2000), their low level of polymorphism precludes using them as sources of markers. To counteract this potential problem, we designed the second primer set with a core containing the AATT sequence near the 3′ region in order to aim at AT-rich regions. Normally these are found more frequently on promoters and introns (Lin et al. 1999; The EU Arabidopsis Genome 1999). Since introns, promoters and spacers are usually variable among different individuals, this intrinsic dissimilarity makes it feasible to generate polymorphic bands based on introns and exons. Why AATT is used in reverse primer:

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Sequence-related amplified polymorphism (SRAP) For the first five cycles the annealing temperature is set at 35°C. The following 35 cycles are run at 50°C. The amplified DNA fragments are separated by denaturing acrylamide gels and detected by Autoradiography. PCR ELECTROPHORESIS

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After sequencing, approximately 45% of the gel-isolated bands matched known genes in the Genbank database. Twenty per cent of the SRAP markers were co-dominant, which was demonstrated by sequencing. Construction of a linkage map revealed an even distribution of the SRAP markers in linkage groups. Sequence-related amplified polymorphism (SRAP) BLAST

Crops where SRAPs have been used : 

Crops where SRAPs have been used Potato Rice Lettuce Chinese cabbage (Brassica rapa L.) Rapeseed (Brassica napus L.) Garlic Apple Citrus Wheat G. Li · C.F. Quiros (✉) Department of Vegetable Crops, University of California, Davis, CA 95616, USA e-mail: cfquiros@ucdavis.edu Tel.: 530-752-1734, Fax: 530-752-9659 Sequence-related amplified polymorphism (SRAP)

Why annealing temperature is changed from 35 to 50 degree C : 

Why annealing temperature is changed from 35 to 50 degree C The initial annealing temperature for the first five cycles was set at 35°C. The rationale behind using this temperature is based on the fact that primer annealing to the DNA template depends on the matching-level of both sequences, and amplification efficiency is determined by the effectiveness of the primer binding. The low initial annealing temperature ensures the binding of both primers to sites with a partial match in the target DNA. The annealing temperature is then increased for the subsequent 35 cycles to 50°C. This temperature change ensures that the DNA products amplified at the first five cycles were efficiently and consistently amplified in exponential fashion during the rest of the cycles. When the annealing temperature was kept at 35°C for all 40 cycles, it resulted in bands of poor reproducibility. Sequence-related amplified polymorphism (SRAP)

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SRAP GEL Sequence-related amplified polymorphism (SRAP)