Susanne Carpenter Lab Notebook

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Dahlquist Lab

Lactase Persistance

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Week 3

June 6, 2019

Goals:

  • Analyze SNP sequence results using Blast2Seq
  • Upload edited text files to Box

Results:

7CC-for-for_edited and 7CC-rev-rev_edited Blast

  • Query cover = 100%
  • Percent identification = 100.00%
  • Query length = 361
  • Max/total score = 667
  • Accession = Query_201569
  • E value = 0.0

7CC-for-for_edited with PCR Sequence BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 361/361 bp
  • Query length = 448
  • Max/total score = 667
  • Accession = Query_61941
  • E value = 0.0

7CC-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 361/361 bp
  • Query length = 448
  • Max/total score = 667
  • Accession = Query_197899
  • E value = 0.0

15TT-for-for_edited and 15TT-rev-rev_edited Blast

  • Query cover = 100%
  • Percent identification = 100.00%
  • Query length = 356
  • Max/total score = 658
  • Accession = Query_228743
  • E value = 0.0

18CT-for-for_edited and 18CT-rev-rev_edited Blast

  • Query cover = 100%
  • Percent identification = 100.00%
  • Query length = 358
  • Max/total score = 662
  • Accession = Query_16877
  • E value = 0.0

Week 4

June 12, 2019

Goals:

  • Analyze SNP sequence results of plasmids using Blast2Seq
  • Upload edited files to Box

Results:

Sequence result pictures

CC1-for-for_edited and CC1-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 537/537 bp
  • Query length = 537
  • Max/total score = 992
  • Accession = Query_44385
  • E value = 0.0

CC1-for-for_edited with PCR Sequence BLAST

  • Query cover = 83%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_61749
  • E value = 0.0

CC1-rev-rev_edited with PCR Sequence

  • Query cover = 83%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_76995
  • E value = 0.0

CC2-for-for_edited and CC2-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 560/560 bp
  • Query length = 560
  • Max/total score = 1035
  • Accession = Query_233573
  • E value = 0.0
    • CC2-for-for_edited and CC2-rev-rev_edited both had a point mutation in their sequence that was different from the other CC samples, therefore it will not be considered for further testing.

CC2-for-for_edited with PCR Sequence BLAST

  • Query cover = 79%
  • Percent identification = 99.33%
    • 445/448 bp
  • Query length = 448
  • Max/total score = 809
  • Accession = Query_6267
  • E value = 0.0

CC2-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 79%
  • Percent identification = 99.33%
    • 445/448 bp
  • Query length = 448
  • Max/total score = 809
  • Accession = Query_121471
  • E value = 0.0

CC3-for-for_edited and CC3-rev-rev_edited

  • CC3-rev-rev most likely had the primer slip off, which caused a majority of the sequence to be Ns, therefore, since the CC3 could not be compared to itself, it will not be considered for further testing.

CC3-for-for_edited with PCR Sequence BLAST

  • Query cover = 82%
  • Percent identification = 100.00%
    • 437/437 bp
  • Query length = 448
  • Max/total score = 808
  • Accession = Query_142855
  • E value = 0.0

CC4-for-for_edited and CC4-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 534/534 bp
  • Query length = 534
  • Max/total score = 987
  • Accession = Query_194449
  • E value = 0.0

CC4-for-for_edited with PCR Sequence BLAST

  • Query cover = 83%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_216223
  • E value = 0.0

CC4-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 83%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_70755
  • E value = 0.0

CC5-for-for_edited and CC5-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 99.65%
    • 564/566 bp
  • Query length = 566
  • Max/total score = 1033
  • Accession = Query_ 36879
  • E value = 0.0

CC5-for-for_edited with PCR Sequence BLAST

  • Query cover = 79%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_216103
  • E value = 0.0

CC5-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 79%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_48199
  • E value = 0.0

TT1-for-for_edited and TT1-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 541/541 bp
  • Query length = 541
  • Max/total score = 1000
  • Accession = Query_200641
  • E value = 0.0

TT1-for-for_edited with PCR Sequence BLAST

  • Query cover = 82%
  • Percent identification = 100.00%
    • 448/448 bp
  • Query length = 448
  • Max/total score = 828
  • Accession = Query_137985
  • E value = 0.0

TT1-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 82%
  • Percent identification = 100.00%
    • 448/448 bp
  • Query length = 448
  • Max/total score = 828
  • Accession = Query_84291
  • E value = 0.0

TT2-for-for_edited and TT2-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 99.82%
    • 554/555 bp
  • Query length = 555
  • Max/total score = 1018
  • Accession = Query_168699
  • E value = 0.0

TT2-for-for_edited with PCR Sequence BLAST

  • Query cover = 80%
  • Percent identification = 98.88%
    • 443/448 bp
  • Query length = 448
  • Max/total score = 798
  • Accession = Query_140379
  • E value = 0.0

TT2-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 80%
  • Percent identification = 98.88%
    • 443/448 bp
  • Query length = 448
  • Max/total score = 798
  • Accession = Query_29783
  • E value = 0.0

TT3-for-for_edited and TT3-rev-rev_edited

  • TT3-rev-rev most likely had the primer slip off, which caused a majority of the sequence to be Ns, therefore, since the TT3 could not be compared to itself, it will not be considered for further testing.

TT3-for-for_edited with PCR Sequence BLAST

  • Query cover = 51%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_236811
  • E value = 0.0

TT4-for-for_edited and TT4-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 553/553 bp
  • Query length = 553
  • Max/total score = 1022
  • Accession = Query_167069
  • E value = 0.0

TT4-for-for_edited with PCR Sequence BLAST

  • Query cover = 81%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_219933
  • E value = 0.0

TT4-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 81%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_238039
  • E value = 0.0

TT5-for-for_edited and TT5-rev-rev_edited BLAST

  • Query cover = 100%
  • Percent identification = 100.00%
    • 563/563 bp
  • Query length = 563
  • Max/total score = 1040
  • Accession = Query_43015
  • E value = 0.0

TT5-for-for_edited with PCR Sequence BLAST

  • Query cover = 79%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_135727
  • E value = 0.0

TT5-rev-rev_edited with PCR Sequence BLAST

  • Query cover = 79%
  • Percent identification = 99.78%
    • 447/448 bp
  • Query length = 448
  • Max/total score = 822
  • Accession = Query_155103
  • E value = 0.0

Week 6

SURP 2019 Abstract Draft

Successful implementation of an RFLP assay to detect the human C/T-13910 SNP which is associated with lactase persistence

Susanne M. Carpenter, Alyssa N. Weisblatt, Kam D. Dahlquist

Human infants, like those of other mammals, express the lactase enzyme to digest the lactose sugar in mother's milk. After weaning, the lactase-phlorizin hydrolase gene (LCT) ceases to produce lactase, resulting in the lactase non-persistence (LNP) phenotype. However, in 35% of the world’s population of humans, expression of the lactase enzyme continues into adulthood, known as lactase persistence (LP). The LP phenotype occurs when an individual has one of more than 20 single nucleotide polymorphisms (SNPs) located near the lactase gene. The most commonly studied SNP is the "European" SNP, C/T-13910, which occurs in an enhancer region upstream of the LCT gene in intron 13 of the neighboring MCM6 gene. The T allele correlates with LP, as it results in a higher expression of lactase due to increased binding of the Oct-1 regulatory transcription factor and/or prevention of gene silencing. Several studies have been conducted in non-European, isolated populations to determine the frequency of LP within the ethnic group. However few studies have been conducted in ethnically diverse locations such as the US. To conduct such a study at Loyola Marymount University, we successfully implemented a restriction fragment length polymorphism (RFLP) assay reported in Morales et al. (2011) to detect the C/T-13910 SNP in human cheek cell lysate, confirming the results by direct sequencing of the DNA. We used the TOPO cloning technique to generate control plasmids with both genotypes to use as template in future RFLP assays. We confirmed that the sequence of the control plasmid inserts matched the original target sequence. Furthermore, we curated a list of SNPs from the literature and public databases that are known to affect the LP phenotype. With this information, we plan to develop additional RFLP assays to test for SNPs commonly found in populations of non-European descent, such as the G/C-14010, T/G-13915, and C/G-13907 variants. During the 2019-2020 academic year we will collect samples from a diverse set of LMU students and compare their genotypes for the C/T-13910 SNP with their self-reported LP or LNP phenotype. Having determined the genotype and phenotype frequencies for a US population, we will explore the implications of this work for the creation of dietary guidelines in the US that are useful for all people.

368 words

Fall 2019

Experimental Biology 2020 Abstract Draft

Development of RFLP and qPCR Assays to Detect the Human C/T-13910 SNP, which is Associated with Lactase Persistence

Susanne M. Carpenter, Alyssa N. Weisblatt, Kam D. Dahlquist

Human infants, like those of other mammals, express the lactase enzyme to digest the lactose sugar in mother's milk. After weaning, the lactase-phlorizin hydrolase gene (LCT) ceases to produce lactase, resulting in the lactase non-persistence (LNP) phenotype. However, in 35% of the world’s population of humans, expression of the lactase enzyme continues into adulthood, known as lactase persistence (LP). The LP phenotype occurs when an individual has one of more than 20 single nucleotide polymorphisms (SNPs) located near the lactase gene. The most commonly studied SNP is the "European" SNP, C/T-13910, which occurs in an enhancer region upstream of the LCT gene in intron 13 of the neighboring MCM6 gene. The T allele correlates with LP, as it results in a higher expression of lactase due to increased binding of the Oct-1 regulatory transcription factor and/or prevention of gene silencing. Several studies have been conducted in non-European, isolated populations to determine the frequency of LP within the ethnic group. However, fewer studies have been conducted in ethnically diverse locations such as the US. To lay the groundwork for such a study, we have successfully implemented a restriction fragment length polymorphism (RFLP) assay reported in Morales et al. (2011, doi:10.1136/bmjopen-2011-000125) to detect the C/T-13910 SNP in human cheek cell lysate, confirming the results by direct sequencing of the DNA. We used the TOPO cloning technique to generate control plasmids containing inserts with either the "C" or "T" genotype to use as positive controls in future assays. We confirmed that the sequence of the control plasmid inserts match the original target sequence. We are using the control plasmids to optimize a SYBR green quantitative polymerase chain reaction (qPCR) genotyping assay based on Weinlander et al. (2010, doi:10.1002/bmb.20357) as an alternate method of genotyping. Finally, we have curated a list of SNPs from the literature and public databases that are known to affect the LP phenotype in different world populations.

Spring 2020

LMU Undergraduate Research Symposium 2020 Abstract Draft

Implementation of RFLP Assay to Detect the Human C/T-13910 SNP, which is Associated with Lactase Persistence

Susanne M. Carpenter, Alyssa N. Weisblatt, Kam D. Dahlquist

Human infants express the lactase enzyme to digest the lactose sugar in mother's milk. Post-weaning, the lactase-phlorizin hydrolase (LCT) gene ceases to produce lactase, resulting in the lactase non-persistence (LNP) phenotype. However, 35% of the world’s population continues to express lactase into adulthood, known as lactase persistence (LP), and is caused by having a single nucleotide polymorphism (SNP) located near the lactase gene. The most commonly studied SNP is the "European" SNP, C/T-13910, which occurs in an enhancer region upstream of the LCT gene in intron 13 of the neighboring MCM6 gene. The T allele correlates with LP, as it results in a higher expression of lactase due to increased binding of the Oct-1 regulatory transcription factor and/or prevention of gene silencing. Few studies have been conducted on LP frequency in ethnically diverse locations such as the US. To lay the groundwork for such a study, we developed a restriction fragment length polymorphism (RFLP) assay reported in Morales et al. (2011) to detect the C/T-13910 SNP in human cheek cell lysate. We used the TOPO cloning technique to generate control plasmids containing inserts with either the "C" or "T" genotype to use as positive controls. We are also developing a genotyping assay using double-mismatch allele-specific quantitative polymerase chain reaction (qPCR; Lefever et al. 2019). This work has implications for the creation of dietary guidelines in the US that are useful for all people.

233 words

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