4 The Genetics Tolerance is a mutation in the LCT gene (Chromosome 2) Everyone has two copes of each gene (maternal and paternal)Each gene has a C or a T at a specific locationThis is called a SNP (single nucleotide polymorphism)TT and TC = tolerantCC = intolerantStudents should read the background information for the lab at this point.What is an SNP? Video
5 The Genetics The individual is lactose TOLERANT T Chromosome 2 from dadTLCTTChromosome 2 from momLCTThe individual is lactose TOLERANT
6 The Genetics The individual is lactose TOLERANT T Chromosome 2 from dadTLCTCChromosome 2 from momLCTThe individual is lactose TOLERANT
7 The Genetics The individual is lactose INTOLERANT C Chromosome 2 from dadCLCTCChromosome 2 from momLCTThe individual is lactose INTOLERANT
8 Where is it most prevalent? Prevalence of lactose tolerance and reliance on dairy products vary throughout the world.
9 Why do we see this pattern? Map shows % intolerance
10 Natural Selection There is variation of traits in a population 2. There is differential reproduction.There is heredity.End result.
11 How can we see this in our genes? PCR (polymerase chain reactionBuild copies of the segment that contains the SNP so we can see itWhat’s in the mix?Master Mix: Taq, dNTPs, bufferPrimer Mix: 4 primers (two outer primers and two inner primers)PCR Reaction VideoThe inner primers tell you your genotype
12 DNA consists of building blocks called nucleotides What is DNA Made Of?DNA consists of building blocks called nucleotidesImage: SCFBIO1) a phosphate moleculegives DNA its negative charge2) a pentose sugarfive-carbon sugar in ring form3) a nitrogenous basering of carbon and nitrogen atomsvariable
13 2 Types of Nitrogenous Bases (4 in total) Pyrimidines: 6-member ringCytosine & ThyminePurines: fused 5 & 6 member ringsAdenine & GuanineA T C G =DNA alphabet
14 The Base Pair Rules Hydrogen bonds form between bases A = T two hydrogen bondsG C three hydrogen bondsThe bonds are weak and can bebroken by high temperatures
15 DNA has two strands with bases paired in the middle
16 DNA ReplicationMolecular Cell Biology, Lodish et. al. 4th ed.
17 Deoxynucleotide-triphosphates: dNTPs The “PCR Building Blocks”A “dNTP mix” contains equal amounts of :dATPdTTPdGTPdCTP11
18 The PCR “Cycle” Separates double helix into two strands Denature: 94-96oC…Anneal: oC…Extension: 72oC…Repeat steps 1-3:Separates double helixinto two strandsPrimers bind to target site onsingle stranded DNADNA polymerase adds dNTPs according to the base pairing rules (polymerization)5 to 40 times using a Thermal Cycler3
19 After 30 cycles, DNA is amplified over a billion-fold! Target sequenceCycle 2Cycle 1
21 Tetra-primer ARMS-PCR Procedure for SNPs Tetra-primer ARMS-PCR is a widely accepted method for amplifying SNP DNA. Unlike the traditional PCR procedure, it employs two sets of primers. The “outer primers” set the boundary for the DNA region containing the SNP. The “inner primers” are specific for each allele. During the PCR procedure, you will obtain either two or three different amplified DNA segments, depending upon genotype.When the tetra-primer ARMS-PCR procedure is completed, the DNA fragments are separated by gel electrophoresis. The PCR products produce a characteristic fragment size pattern: Heterozygous individuals have one non-specific band and two specific bands of known length (3 bands total). Homozygotes have one non-specific and one specific band (two bands total).A non-specific DNA fragment is produced in all all individuals. This DNA fragment results from the outer primers and serves as an internal positive control for the PCR procedure.Homozygotes will produce one additional fragment resulting from one of the inner primers.Heterozygotes will produce two additional DNA fragments, one from each of the inner primers.The inner and outer primer arrangement for one particular SNP is shown below. The outer primer pair allows for the non-specific band to be produced. Each inner primer (G or A) is allele-specific, and individuals will produce that DNA segment only if they carry the specific allele.
23 Agarose Gel Electrophoresis DNA“Ladder”well#1#2#3+_large DNAfragmentssmall DNADye is added togive the DNA colorElectron micrograph of an agarose gel
24 The Tetra-Primer System It’s not super “clean”You get primer dimersInner primers are similar and stick togetherIgnore them on the gel (they’re smaller than 100 bp)The outer primers are sometimes non-specificThe fragments are small! Run the gel for the full time to separate out the bands!
25 Sample Gel: Focus on the “lac” Lane This gel shows some of the issues that might be seen with use of the tetra-primer system
26 How to Interpret the Gel Extraneous bands at 400, 500Outer band- 268Inner band: 188Inner band: 135 (faint)Primer dimers <100