Genotypic Identification of STK11 homologs in Homo sapiens and Daphnia pulex using PCR

Andrew Ingersoll, Brian Snyder, and Ali Gabrion

Abstract

Germline mutations of the tumor suppressor gene Serine/threonine kinase 11 (STK11), also known as liver kinase B1 (LKB1), are responsible for Peutz-Jeghers syndrome (PJS) an autosomal dominant-inherited disease characterized by gastrointestinal polyposis, mucocutaneous pigmentation and cancer predisposition (Su et. al., 1999). Our purpose is to identify published primer sequences and use them to detect and replicate the STK11 gene in humans (Homo sapiens) using polymerase chain reaction (PCR) amplification and gel electrophoresis. We also plan to detect and replicate the SKb11 gene, an STK11 homolog, in Daphnia pulex (common water flea) using PCR and designed primers. We hypothesize that we can amplify the STK11 gene in humans from bronchial epithelial cells from the IB3-1 cell line using PCR and published primers. We also hypothesize that we can amplify the STb11 gene using genomic DNA from Daphnia pulex using PCR and designed primers because the primers match to the DNA template and will anneal to it (Connolly et. al, 2000; Abdellah et.al., 2004). Genomic DNA from human bronchial epithelial cells was extracted and purified from culture using the “Generation Capture Column” kit from Qiagen Inc. Genomic DNA from Daphnia pulex was purified from Daphnia pulex using a squishing buffer and the “Generation Capture Column” kit from Qiagen Inc. By process of hot-start PCR, DNA templates from humans as well as Daphnia pulex were amplified using published and designed primers, respectfully. The PCR product was analyzed using gel electrophoresis. A PCR product of 240 base pairs was formed from the published primers which anneal at base pairs 38,545 and 38,785 of the human STK11 gene (Connolly et. al., 2000; Abdellah et.al., 2004). A PCR product of 737 base pairs was formed from the designed primers and Daphnia pulex DNA because the primers anneal at base pairs 242,362 and 243,099 of the Daphnia pulex SKb11 gene (Colbourne et. al., 2011). PCR analysis of DNA is useful in determining genotype as well as determining gene sequence information. Our results will help identify a possible homolog to the STK11 gene in Daphnia pulex as well as confirm the research done by other scientists, by testing their primer sequences.

Discussion

Peutz-Jeghers syndrome, an autosomal dominant disease, results from a mutation in one allele of the STK11 gene to cause the gene to produce a nonfunctional protein. The STK11 gene encodes the LKB1 tumor suppressor protein (Hemminki et. al., 1998). The LKB1 protein is responsible for regulating cellular proliferation by controlling high energy processes such as cellular division (Sanchez-Cespedes, 2007). We know that that the STK11 gene will be identified in humans as well as its homolog, SKb11 in Daphnia pulex and amplified using PCR that has been designed using specific primers and experimentally optimized conditions such as annealing temperatures and buffer concentrations.

We predict that using published primers and primers of our own design, that we can amplify the wild type STK11 gene using genomic DNA from human bronchial epithelial cells as a template, because the primers will be successful in annealing to the targeted sequences and through extension, allowing for successful recreation of the targeted sequence (Garibyan et. al., 2013). We also predict that the STK11 homolog, SKb11 in Daphnia pulex can also be amplified using PCR and identical primer sequences to the ones that we designed for human genomic DNA. We predict that gel electrophoresis will produce a band at 737 base pairs, because the published primers target 737 base pairs between base pairs 242,362 and 243,099 (Connolly et. al., 2000) of the Daphnia pulex genome.

All primers have been checked using BLAST to make sure that non-specific binding is not a problem. In each case, the primers only match to their complementary sequence once, with that spot being the desired location. We predict that the annealing temperature for We predict that both results will support our hypothesis as we are able to show that the STK11 gene is present in humans and its homolog, SKb11 is present in Daphnia pulex.

Future Directions: Many problems arose when attempting to amplify E. coli DNA for the first part of the project. The first few trials resulted in no amplification. Further research into PCR resulted in us adding MgSO₄ and higher concentrations of DNA and primers. This caused successful amplification of the E. coli DNA. Although the PCR buffer that we used contained MgSO₄, it is probable that the concentration was not high enough to cause successful amplification. We also used longer PCR cycles at lower annealing temperatures to cause better amplification. Primers will not anneal if the temperature is too high, but they will still anneal at lower temperatures, and we believe that this helped solve our problems. For the Daphnia pulex and human PCR, we used higher concentrations of MgSO₄ and lower temperatures immediately, and this resulted in successful amplification on the first try.

Figure 2. (a) Gel picture using published primers and DNA from human bronchial epithelial cells, cell line IB3-1, to amplify exon 8 of the STK11 gene. Invitrogen 100 base pair DNA ladder with known band lengths marked is shown in wells #1 and #6. Wells marked #2 and #3 had an annealing temperature of 60°C and wells #4 and #5 had an annealing temperature of 64°C. Wells #7 and #8 contained negative controls with #7 run under the same conditions as #2 and #4, and #8 run under the same conditions as #3 and #5. The published primers have sequences: forward 5’-CCTGACAGGCGCCACTGCTTC-3’ and reverse 5’-GGCCCCCCGCCAGACTCAC-3’. The primers targeted a 240 base pair long sequence. The PCR reaction mixture contained the extracted sample of human bronchial epithelial cell DNA, a 1X PCR buffer, 100 umol/L dNTPs, 0.1 umol/L of the reverse primer, 0.1 umol/L forward primer, 1.5 mM MgSO₄ and 2.5 U Taq polymerase. Wells number #3, #5, and #8 had the same reaction mixture except with 3 mm/L MgSO₄ and 0.2 umol/L of both forward and reverse primers. The sample was then placed into wells on an agarose gel containing 40 mL 1x LB, 0.4 g agarose, and 1 uL of GloGreen loading dye. The PCR cycle started with 10 minutes at 95°C for initial denaturation, then 42 cycles of 30 seconds at 95°C, 30 seconds at 60°C or 64°C, and 30 seconds at 72°C, followed by a final extension of 5 minutes at 72°C. A fragment of 240 base pairs was found (Connolly et. al., 2000; Abdellah et.al., 2004). (b) Semi-log plot made using Invitrogen 100 base pair DNA ladder. The points on the graph denote the distance traveled by each DNA band of known size. A logarithmic trend line was added, with equation shown. The red lines indicate the average distance traveled and band size of the bands shown in (a). An R² value of 0.9844 was obtained for the trend line, suggesting that the trend line is very accurate. The equation of the trend line was used to calculate the size of the bands from PCR from the distance that our bands traveled down the gel. Our bands traveled an average of 4.08 cm. An average value of 237.56 base pairs was obtained for the bands shown in (a). The actually size should be 240, so this suggests that the correct PCR product was formed and that the DNA was correctly amplified.