FTB2024 Lab Lecture (PDF)

How can you taste bitter? Dr Pam Scott Course email lifesci-fundamentals-of-biology2@gl asgow.ac.uk  Aims of the lecture….. To understand how the labs will be delivered in FTiB; To appreciate how we taste bitter chemicals in our foods; To understand the SNPs involved in...

How can you taste bitter? Dr Pam Scott Course email lifesci-fundamentals-of-biology2@gl asgow.ac.uk  Aims of the lecture….. To understand how the labs will be delivered in FTiB; To appreciate how we taste bitter chemicals in our foods; To understand the SNPs involved in the bitter taste perception gene (TAS2R38); To describe the methods used to determine genotype of bitter taste perception.  Lab help and support….. Your peers in the labs; Moodle forum; R help sessions; Jenny August – SLD Maths Adviser; Attend face-to-face labs.  Health and Safety in the Lab…. Dress code: lab coat, no shorts, no sandals, tie hair back, safety glasses; Wash hands coming into/going out of the lab; No eating or drinking; Put coats/bags under the bench; Keep your bench area clean.  Overall aim of the lab course….. To characterize phenotypic and genotypic variation for sensory perception of the bitter taste in a student population. w to prepare buffer solutions; w and why restriction enzymes are used in molecular biology w PCR works and how it is used in molecular biology labs; hat SNPs are and why they are important; w gel electrophoresis can be used to analyse DNA; w to analyse data sets; w to present the results of experiments; w to use R to perform statistical analysis. Labs 1-6 …..  Practical Skills monitoring in 2X GTA observes a skill during the lab This doesn’t change what you do in the lab Multiple opportunities to practice and demonstrate your skill development Checklist on Moodle shows your practical skills development over your degree Achievement appears on your HEAR transcript  Practical Skills monitoring in 2X There will be six skills checked over the six labs: 1. Pipetting 2. DNA gel loading 3. Changing the pH of a solution 4. Using a centrifuge 5. Data analysis using R 6. Creating a professional LinkedIn What is PTC? Phenylthiocarbimide Has a similar chemical group found in Brassica vegetables; PTC tastes bitter to ~70% of the population. Why can only some people taste PTC? TAS2R3 8  Bitter taste Discovery The inability to taste phenylthiocarbamide (PTC) is a genetic trait that varies in the human population. Bitter Taste Process  Bitter Taste Process TAS2R38 encodes for a protein receptor that mediates the perception of bitterness through a G protein-coupled second messenger pathway ; Variations in the gene sequence leads to ‘non taster’ or ‘taster’ phenotypes; Sequencing identified three nucleotide positions of TAS2R38  Bitter Taste Perception PositionSNPs in the TAS2R38 Taster Gene (T) Non-taster (t) 145 C (proline- G (alanine - P) A) 785 C (alanine - T (valine - V) A) 886 G (valine - A (isoleucine TT – homozygous V) dominant – strong - I) taster tt – homozygous recessive – non-taster Tt – heterozygous - weak taster  Overview of Experimental Plan 312 bp 312 bp 206bp 312 bp 106bp  Polymerase Chain Reaction Genomic DNA Gene of Interest Amplified DNA (buccal swab) (TAS2R38) (billions of copies)  Polymerase Chain Reaction Repeated 25-30 times  What goes into a PCR? Template to be amplified (genomic DNA from buccal swab) Forward and reverse primers Taq DNA polymerase dNTPs Mastermix Buffer to maintain pH and provide Mg2+  Amplifying TAS3R38 Using PCR PCR primers amplify 312bp region of TAS2R38 gene Left Chromosome Primer 7 TAS2R38 gene (1143 Right bp) Primer Amplified region of 312 bp contains SNP 785...TGTGCTGCCT.....TGTGTTGCCT.....ACACGACGGA......ACACAACGGA taster allele Non-taster allele  Restriction Endonucleases Restriction enzymes cut DNA at a SPECIFIC nucleotide sequence called a restriction site The Fnu4H1 restriction enzyme uses GCNGC recognition site – It only digests the TAS2R38 gene at this site Remember the TAS2R38 gene contains single nucleotide polymorphisms – You may be GCTGC and/or GTTGC at position 785 – The Fnu4H1 may or may not digest the DNA in the given samples  Restriction Digest of SNP 785 with Fnu4H1 PCR PRODUCT = 312 BP length 785 SNP...TGTGCTGCCT.....TGTGTTGCCT......ACACGACGGA......ACACAACGGA.. taster allele Non-taster allele Add Fnu4H1 for restriction digest...TGTGC TGCCT......TGTGTTGCCT.....ACACGA CGGA.......ACACAACGGA.. 2 fragments 1 fragment 206bp 106 bp 312bp Genotype Analysis wells 312 bp 206 bp 106 bp tt TT  Lab Report To determine whether there is a link between the PTC taster genotype and variables of your choice​from a data set provided to you Abstract – should summarise your findings Introduction​- should include the purpose of experiments and a bit of background to the topic Methods​- lab analysis of the gene and statistical approach Results​ - appropriate well annotated figures and tables with legends​ - a main text which summarises what was seen in each figure Discussion​ References (Harvard style)​  Science News Essay 500 words +/- 10% (figure legends don’t count); Deadline 14th Oct – Turnitin; Look at "Writing Guide for the Science News Essay" and "How Good is your Marking" on Moodle; Find a 2024 science news item eg from Science Daily (link this in the introduction of the essay); Search in the news item for the original ‘peer reviewed’ article – must be from 2023 or 2024; Research article should have a molecular link; Tips: include a figure or two (don’t plagiarise); don’t choose a meta- analysis paper that is stats heavy; think about the balance of the essay.