Genetics module Lectures DNA Structure, Replication The Genetic Code; Transcription and Translation Principles of Heredity; Gene Mapping Controlling Gene Expression Mutation and Cancer Textbook: Introduction to Genetic Analysis, Griffiths et al., 9th Ed. 1
Practicals Manipulating DNA: - Extracting DNA from Cells - Polymerase Chain Reaction - Restriction Enzyme Digestion - Gel Electrophoresis Studying Effects of Genetic Variation: - A Matter of Taste Analysing Sequences: - DNA, RNA, Protein Analysing Gene Expression: - Comparing Expression in Human Tissues - Transgenic Reporter Genes Assessment Lab-book reports (30%) Multiple choice exam on Day 5 (70%) Lab-book reports: - Aim - Observations - Discussion Due on Days 2, 3, 5 2
Genetic differences in taste perception 3
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Different tastes detected by different types of receptor proteins Expressed in different cells in taste buds 5
Taste receptor genes Single genes for sweet, umami, sour receptors Large family of bitter receptor genes - different ones detect different chemicals - all transmit same signal: bitter Species-specificity - repertoire of receptors adapted for chemicals that must be detected e.g. cats lack functional sweet receptor and do not prefer sweet-tasting foods Genetic variation within species - mutations in taste receptor genes account for some differences in people s tastes 6
TAS2R38 receptor detects PTC Phenylthiocarbamide But only some people can taste PTC! This difference is inherited Due to mutations in TAS2R38 gene 7
Cloning the PTC taste sensitivity gene Taster phenotype found to run in families Transmitted as a Mendelian trait - one major gene involved Gene was mapped to chromosome 7 Mutations in particular gene segregate with phenotype TAS2R38 gene: encodes a bitter receptor protein Red: Taster sequence Blue: Non-taster sequence 8
Two major alleles (variants) in human population: T allele: ~40% (Taster, CCA codes for Proline at position 145) N allele: ~60% (Non-taster, GCA codes for Alanine at position 145) Only need one functional (taster) allele to be able to taste PTC What is expected frequency of tasters and non-tasters in the population? Can you think of another case where a mutation in a receptor protein alters perception? Determining which TAS2R38 alleles you carry (your genotype) Isolate some of your DNA How to analyse just the TAS2R38 gene? (among 3x10 9 total bases of sequence) Use Polymerase Chain Reaction to amplify the TAS2R38 gene sequence specifically How to tell which allele(s) you have? Use Restriction Enzyme that digests one allele but not the other 9
Isolating your DNA Collect some cells: - rinse mouth vigorously with saline to collect cheek cells - spin down saline to concentrate cells Burst them open: - resuspend cells and add small amount to tube containing Chelex resin (binds heavy metal ions that could damage DNA) - boil for ten minutes (bursts cells, degrades proteins) Purify the DNA away from everything else (proteins, lipids) - spin down cell debris and resin - carefully remove supernatant containing DNA Make sure tubes are labeled with your ID number! The Polymerase Chain Reaction Selectively replicate specific sequence over and over (amplification) DNA replication requires: - DNA template - DNA polymerase enzyme - Nucleotides - Primer Primer is a short stretch of DNA that binds to its complementary sequence in the template and acts as start of new strand of DNA Use primers complementary to sequence of interest - amplify only that region of DNA 10
PCR is hugely sensitive Used in forensics to amplify DNA from tiny amounts of cellular material Amplify regions that are highly variable in population: DNA fingerprinting Very sensitive to contamination! 11
Red: Taster sequence Blue: Non-taster sequence Amplifying TAS2R38 sequence from your purified DNA Put some of your purified DNA in PCR tube Use another tube for negative control (no DNA) Add primers,nucleotides, Taq DNA polymerase, buffer Mix together, store on ice, then place in thermal cycler - goes through many rounds of temperature shifts 12
We will use PCR to amplify region of TAS2R38 gene that has polymorphism known to affect ability to taste PTC End up with tube full of just this little stretch of DNA How to tell which allele(s) are present? 1. Could sequence the DNA OR: 2. Use enzyme that can tell the difference 13
Restriction Enzymes Derived from bacteria - host defense versus viruses Restriction Enzymes cleave DNA at a specific sequence (e.g., 5 GAATTC 3, 5 CATATG 3, 5 CTGCAG 3 ) Can you see anything unusual about these sequences? Why don t they cleave the bacteria s DNA? We want an enzyme that will cleave one allele of the TAS2R38 sequence but not the other - need polymorphism itself to be part of the cleavage site HaeIII cleaves sequence: GGCC 14
Gel Electrophoresis How to tell if amplified DNA has been cut by restriction enzyme? Run it through a gel using an electric current Gel matrix retards passage of larger molecules more than smaller ones Separate pieces of DNA by size - visualise with fluorescent chemical label 15
Homozygous N/N Heterozygous T/N Homozygous T/T Control Lane Testing for perception of PTC Does your phenotype correlate with your genotype? Does it correlate with whether you like brussel sprouts? 16
Site GGCC/GGGC not actually there in the genome (usual sequence either AGCC or AGGC) We can change the A to a G in the amplified DNA by incorporating a G at that position in the primer 17