Part:BBa_K3389000

Beta casein exon 7 A2 sequence

This part codes for the exon 7 portion of the A2 allele for the beta casein protein. The A2 sequence variant of beta-casein differs from the more common A1 allele at a single nucleotide position (position 74, A1 has an A at this position, A2 has a C). This results in a proline at position 67 in A2 beta casein protein rather than the histidine found in the A1 protein. The purpose of this part is to serve as positive control DNA for an A2 allele detection system.

Characterization

SUNY_Oneonta iGEM 2020

One purpose of this part is to provide a template for amplification of the A2 version of exon 7 of the beta casein gene via RPA. After initial primer design and analysis of the properties of the proposed oligonucleotides using bioinformatics, we selected three forward primers: 2F, 4F, 18F; and three reverse primers: 6R, 10R, 11R, for testing (Figure 1).  

Figure 1: Primers selected for use in RPA Amplification

We characterized the ability of this part to be amplified via RPA as a function of primer pair sequence and temperature. We began optimizing primer selection by pairing all the forward primers with all possible reverse primers; the purpose of this was to see which primer pairs worked the most effectively together. We then performed RPA using the TwistDx kit, using a 40-minute amplification, performed at 37 °C.  We observed RPA product bands at the predicted size for several primer combinations (Figure 2). Based on the expected amplicon lengths and strength of bands, we decided to choose primer pairs 2F:10R, and 4F:11R as candidates for further optimization of the RPA protocol. 

Figure 2: RPA primer pair optimization. Combinations of forward and reverse RPA primers were used to perform RPA on A2 exon 7 positive control DNA. RPA products were subjected to agarose gel electrophoresis.

We then worked to determine which reaction conditions were ideal for the optimal primer pairings. Each set was tested under the same reaction conditions with the exception of reaction temperature.  We performed reactions for each primer set at 37°C and at 42°C (Figure3). We determined that pair 2F, 10R works best at 37°C and pair 4F, 11R works well at both temperatures.  In the case of primer pair 4F, 11R, we observed additional bands of a higher than predicted molecular weight in the 37°C reaction.

Figure 3. RPA temperature optimization. RPA reactions using the indicated primer pairs were run at 37°C or 42 °C for 40 minutes and RPA products were analyzed via agarose gel electrophoresis.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]