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Genes for Speed or Endurance? Muscle power Skeletal muscles are made of long cells called muscle fibres. There are two types of muscle fibres, slow twitch or muscle contraction and fast twitch. Fast twitch fibres fire more rapidly with more force than slow twitch fibres. Slow twitch fibres are more efficient in using oxygen to generate energy, while fast twitch fibres are less efficient in energy generation. Genetics The DNA molecule is the carrier of genetic information. Genes consist of the four types of DNA building bases called A, C, G, & T. The order of these bases on the chromosomes codes for assembling the order of amino acids to make a protein. Proteins make cells. Athletic performance is built upon good genetics, nutrition, training and mental traits. DNA testing can now be used to profile genetic variations that are associated with power or endurance. The identification of suitability genes in young athletes might enable better decision-making about which sport their physiology equips them for. The relative contributions of training and genetics to success seem to vary depending on the sport and the person but some companies are now offering the tests. ACTN3 in muscle fibre Mutation in ACTN3 gene The major protein components of skeletal muscle are actin and myosin. Actin filaments are stabilized by actin binding proteins or actinins of which there are two types, 2 and 3. Specific genes, ACTN2 and ACTN3, code for these actinin proteins respectively. ACTN3 is expressed only in fast twitch fibers A mutant form of the ACTN3 gene has a single base substitution in the DNA and is referred to as R577X or X. This mutation (R577X) inserts a premature stop codon into the gene for actinin 3 and does not produce the protein. People with this gene do not produce actinin 3 in their fast twitch fibers. 1 What does this DNA mutation mean? No disease is associated with this genetic mutation. Around 16% of the world population is predicted to have the congenital deficiency of actinin-3. Scientists think variations in this gene evolved in response to energy needs of people in different parts of the world during human evolution. Studies have linked the fiber twitch type (fast / slow) with ACTN3, i.e. fast twitch fiber abundant individuals carry the normal version R of ACTN3 and this version is associated with sprint performance while the mutant or non-functional version X is associated with endurance. As people have two copies of each gene they can be RR, RX or XX for the ACTN3 gene. Research has suggested an association between an individual's ACTN3 genotype and athletic performance: R/R genotype: power advantage X/X genotype: endurance advantage R/X genotype: contributes to both power and endurance 2 Testing for ACTN3 The scenario: To test seven young athletes for their ACTN3 genes Your task: To use PCR to identify the ACTN3 genotype of the seven DNA samples. Method: PCR reaction (done before the lab) PCR is used to copy small pieces of DNA in the test-tube. 1. Components were added as in the table below and put into PCR machine for 30 cycles. Tube 1 Tube Tube Tube Tube Tube Tube Tube 2 3 4 5 6 7 8 Mastermix (contains primers, 13µL 13µL 13µL 13µL 13µL 13µL 13µL 13µL dNTPS, MgCL 2 , buffer) 1. Control R/X 1µL 2. Test A DNA 1µL 3. Test B DNA 1µL 4. Test C DNA 1µL 5. Test D DNA 1µL 6. Test E DNA 1µL 7. Test F DNA 1µL 8. Test G DNA 1µL Taq polymerase enzyme 1µL 1µL 1µL 1µL 1µL 1µL 1µL 1µL 2. These tubes were put into the PCR machine to heat and cool to 95°C, 55°C and 74°C for 30 cycles. The enzyme Taq will copy the section of the ACTN3 gene for each person. To detect the X mutation the PCR fragments are then cut with the Dde I restriction enzyme. 3. The R (normal ACTN3 gene) gives one band and the X (mutant ACTN3 gene) gives two bands due to the presence of the Dde I cutting site. 4. The PCR and restriction digests have been done for you. You will separate the bands on a DNA electrophoresis gel in the lab today. 5. DNA electrophoresis to separate the bands from the PCR: Pipette the samples into the wells of the agar gel in the order indicated below. Run the gels to separate the samples. Take a photo of the result. 1. Control R/X 2. Test A 3. Test B 4. Test C 5. Test D 6. Test E 7. Test F 8. Test G 3 AFTER THE LAB: Decide on the ACTN3 genotype from your gel results and write it down. Note ideas on whether this type of testing is useful Note ideas on any issues it may raise FOR INTEREST: What is polymerase chain reaction (PCR)? PCR copies defined pieces of DNA sequence so there is enough to study in the laboratory. Two short DNA primers define the piece copied – these mark the start and end of the piece. The copying is done by Taq polymerase – an enzyme that joins the A, G, T& C’s according to the template sequence of the sample DNA. Temperature is used to control the reaction: ♦ 95°C first separates the two strands of DNA ♦ 55°C the primers bind to each strand ♦ 74°C Taq enzyme adds bases to the primer following the sample DNA template This pattern of heating & cooling repeats for 30 cycles = billion fold copying of the DNA segment 4