BICD 123 Spring 2007 Long Using PCR based markers to map a mutation in Arabidopsis Introduction In the next two lab courses you ll use a PCR based method to map a developmental mutant in Arabidopsis You ll take advantage of the fact that there are several ecotypes of Arabidopsis that have been isolated from different parts of the world and therefore contain nucleotide differences in their genomes Most of these differences are in noncoding regions and therefore do not affect the function of any genes These differences can be single base pair substitutions are small insetion deletions indels in repetitive sequences These indels can be visualized by designing primers flanking the indel and then performing a PCR reaction that will give a different size product from each ecotype To take advantage of these differences to map a mutant you have to cross your mutant into a different ecotype You then allow this F1 plant to go through meiosis and self During the meiosis the chromosomes from the two ecotypes recombine and give you a mixture of chromosomes in the F2 plants These recombinant F2 plants are your mapping population From your F2 population you select plants that show your original mutant phenotype to isolate DNA Since you know the ecotype that you originally isolated your mutant in you know that at that locus all of the DNA must be from that original ecotype At other loci such as on other chromosomes the DNA can come from either ecotype Another nifty outcome of this meiosis is that DNA around your original locus will also be from the ecotype your mutant came from This is referred to as linkage and will allow you to get a map position for your mutant You ll be doing DNA preps on a F2 population segregating the topless 1 mutant This F2 line was generated by crossing a homozygous tpl 1 plant that originated in the Landsberg erecta Ler background to a wild type Columbia Col plant Once you have DNA you and your labmates will test three sets of PCR primers that detect three indels between Ler and Col Two of these indels are linked to tpl 1 whereas one is not It will be up to you to figure out where tpl 1 maps and where to design your next marker Experimental Plan Day 1 DNA extraction Extract DNA from tpl 1 mutant plants 1 Harvest a whole tpl 1 seedling from the plate provided and place in a numbered 1 5 ml tube Grind the seedling with a disposable pestle for roughly 30 seconds or until no whole tissue is present 2 Add 400 l of extraction buffer and vortex the sample for 5 seconds You can leave this sample at room temperature 3 Centrifuge the extracts for 1 minute at 13 000 rpm full speed in the microfuge 4 Remove 300 l of the supernatant liquid and transfer it to a fresh microfuge tube Make sure to number the new tube Add 300 l of isopropanol mix by inverting the tube and leave at room temperature for 2 5 min 5 Centrifuge again at 13 000 rpm for 5 min in the microfuge Carefully remove the supernatent liquid leaving the pellet in the bottom of the tube 6 Rinse the pellet with 200 l of 70 ethanol Spin for 5 min at full speed Remove the supernatent again leaving the pellet in the bottom of the tube 7 Allow pellet to air dry 15 20 minutes at room temp inverted on a Kimwipe paper towel Make sure it is dry 8 Resuspend the pellet in 25 l sterile water sufficient for a 25 l PCR reaction 2 2 l of this sample is PCR reactions In order to get a rough map position the mapping primers must be done on the same DNA Therefore each group will do 6 PCR reactions Make up your mastermix and then the class will share the DNA preps Methods All of the following steps have to be done on ice 1 Make a PCR Master Mix Reagents H2O 10X Buffer with Mg2 Map primer F Map primer R dNTPs Enzyme Taq Polymerase Total 1 in 1 5 ml Tube 1 reaction l 17 0 2 5 0 5 0 5 2 0 0 5 Master Mix 8X l 136 20 4 4 16 4 23 0 184 2 Aliquot 23 l of Master Mix into 7 PCR tubes 2 3 Label each tube with the primer number and the DNA number 4 Vortex DNA and add 2 l of each DNA sample into each individual tube to make a final volume of 25 l Put the tubes in PCR machine and set the PCR condition Because the only difference between each reaction is the DNA sample we can make the mix that contains everything except DNA first 2 Use special thin wall tube for doing PCR 1 3 PCR conditions Steps Initial Denaturation Denaturation Annealing Extension Final Extension Hold Temperature 94 94 55 72 72 4 Time min 5 0 5 0 5 1 0 5 overnight Repeat 49 times After the PCR reactions are done the TAs will store the samples in the fridge You will analyze the samples in the next lab class Solution DNA extraction buffer Tris HCl pH 7 5 NaCl EDTA SDS 200 mM 250 mM 25 mM 0 5 4