Procedure

= Procedure for the DNA extraction =

 //**Click here for the specifics of the DNA extraction process.**//

**__//What is PCR?//__**

Click here for background information on PCR.

Click here for a paper outlining the PCR process.

**__//Primers//__**

//ITS1 (5¹TCCGTAGGTGAACCTGCGG3¹)//

//ITS4 (5¹TCCTCCGCTTATTGATATGC3¹) both by White et al. (1990).//

**__//The ITS Region//__**

ITS stands for **Internal Transcribed Spacer**, which is a region in all lichen species that is usually amplified to highlight differences between different species of lichens. The ITS region is genetically diverse, thus making it ideal to help us understand and barcode the different species with the ITS region of the DNA. Highlighting and isolating specific regions of DNA allows us to examine a smaller portion of the DNA rather than the whole strand. For example, isolating a few hundred base pairs as opposed to millions of base pairs that exist in the DNA.

The ITS region that we are amplifying is:

61 acgaggaatc cctagtaagc acaagtcatt agcttgtgct gattacgtcc ctgccctttg 121 tacacaccgc ccgtcgctac taccgaatga atggcttagt gaggccctcg aattgaggtt 181 cggagactgg caacagtttc tgttctttga gaagttggtc aaacttggtc atttagagga 241 agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcatt accaatttta <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">301 gcgagcctgg cattacgtcc atggtctcgt gaaattattg tatttaaaac ccacacttct <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">361 ataataaaat aacatgtatc atgataaaaa attaaagatc actttcaaca acggatctct <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">421 tggctctcgc atcgatgaag aacgtagcga agtgcgataa gtagtgtgaa ttgcagaatt <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">481 ccgtgaatca tcgaatcttt gaacgcaaat tgcactccct ggtattccgg ggagtatgcc <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">541 tgtttgaggg tcagtttaaa taaaattcgc tgtgttgctt tttgtaacgc ctcggatttg <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">601 ggttatcttg gcctttgggt taagagacct aaaattgact ttttttgtgc atttttatag <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">661 acgtacataa gatatttttt tattcgtctt ggaatgccaa atttattagg tatgatcatg <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">721 ttgtgtgatt tcgtacctat aggtttcatc atctgacctc aaatcaggta gggacacccg <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;"> 781 ctgaacttaa <span class="Normal__Char" style="background-color: #ffff00; font-size: 8pt;">gcatatcaat aagcggagga aaagaaaata acaatgattc ccctagtaac <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">841 tgcgagtgaa gagggacaag ctcaaatttt aaatctgtta ggtatcacct ggccgagttg <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">901 taatttgaag aagcgttttc tgcgcccttg atcagcccaa gtcctattga atgaggcatc <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">961 atggagggtg ataatcccgt ccggttgatc aatgcgtttt acgatacgct ttcaaagagt <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1021 caagttgttt gggattgcag cttaaaatgg gaggtaaatt tctcctaagg ctaaatattg <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1081 gcgagagacc gatagcgaac aagtaccgtg agggaaagat gaaaagaact ttgaaaagag <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1141 agtaaaatag tgcgtgaaat tgttgaaagg gaaacgattg aagtcagtcg taccgatgga <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1201 caatcagcct tttgagtgcg attctgtggg gcatgaggag gaaaccctcg tgttttgcat <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1261 atttgtgctc tttgggtgta tttgttcgta tggtgggtta acatcaattt tggtcgtcat <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1321 aaaatgacgg gaggaatgta gctttggtct cgtattgaag tgttatagcc tctggtagat <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1381 gtgacgaccg ggattgagga ttgcaacgga tacccctcgg ggctactcgc ctgatctctg <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1441 atcgttactc tggtgccgaa agcttgctta tgtcatcaaa gttgatggtc aaaaggttag <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1501 aacgggttaa attcgttaag gatgttgacg taatggcttt aaacgacccg tcttgaaaca <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1561 cggaccaagg agtctaacat atatgcgagt gttcgggtga gcaaaccctt acgcgtaatg <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1621 aaagtaattt aggtgggaac attttttgtg caccatcgac cgatcaggat aatttttgat <span style="font-family: Georgia,serif; font-size: 11px; line-height: 16px;">1681 agatttgagt aagagca

<span style="font-family: Georgia,serif;">In order to find the area of the lichen DNA that we need to amplify, we used the primer found by White et al (Duke University; primer found in 1990) for ITS1 and ITS4. We know that the primer ITS1 ( //<span style="font-family: Georgia,serif;">5¹TCCGTAGGTGAACCTGCGG3¹) //<span style="font-family: Georgia,serif;">will bind to the start of the sequence and isolate downwards on the DNA and that ITS4 //<span style="font-family: Georgia,serif;">(5¹TCCTCCGCTTATTGATATGC3¹) // <span style="font-family: Georgia,serif; font-size: 120%;">provides the termination of the sequence because it attaches to the other strand of DNA. Then the ITS 1 and works in the opposite direction, thus highlighting a specific region.

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