How will DNA help us answer questions about lobster diversity?
To answer my research questions regarding diversity of host and parasite, I will need to be able to reliably determine the identity of both associates. Traditionally, morphological (physical) and chemical traits were used to identify fungal species. However, with the ability to isolate and sequence DNA, scientists now have a much more consistent method of assigning specimens to species.
DNA sequences are considered a more reliable means of identifying species for many reasons. For one thing, choosing morphological characters that are a useful diagnostic for defining species (especially fungal species) can be really hard. It's difficult to know whether all members of a species have a trait, or if a trait is shared with other species, so the trait may not be a defining characteristic. There also can be subjectivity in how different people measure the trait.
In contrast, a DNA sequence can act as a unique "barcode" hundreds of characters long and is read reliably through sequencing. When the right sequence of DNA is selected from a region in the genome, its order of nucleotide bases (A, T, G, C) should vary between species, but not within species. With each base acting as a character, DNA barcoding can also give us information about how closely related species are to each other, because more closely related species should have more similar barcoding regions. (However, to get a more accurate family tree, multiple regions of the genome must be sequenced and compared.) The common barcoding region for fungi (also frequently used for plants) is the ITS region, or the Internal Transcribed Spacer region. This stretch of DNA sits between genes which code for different parts of the ribosome.
So, I'll be using DNA barcoding to identify the species of host and parasite in the lobster mushroom association. I can also build family trees, or phylogenies, to determine whether specialization may have evolved in the parasite--if different branches of the parasite phylogeny are found on different hosts, this could imply that they have evolved a preference for certain hosts.
To obtain my DNA from dried samples, I'll use an extraction procedure that lyses (breaks open) cells so that the DNA is exposed. Some extraction procedures include steps for filtering out other unwanted cellular components like proteins that may degrade DNA. Next, I'll need to copy the extracted DNA using polymerase chain reaction (PCR). PCR uses an enzyme called DNA polymerase, which copies the DNA in our bodies during mitosis and meiosis. PCR has many cycles of different temperates which break double stranded DNA apart into single strands (high temp), allow short pieces of DNA complimentary to the region being copied called primers to anneal to the single strands (lower temp), and activate the DNA polymerase (medium temp). These three steps occur over and over again, doubling the amount of DNA in each cycle.
Once the DNA has been copied through PCR, it will be cleaned and prepared for sequencing. My lab sends out our samples to the sequencing core at our university. They have machines which perform Sanger sequencing, a process that uses fluorescently tagged nucleotide bases to determine the sequence of bases in my sample. Once the samples have been sequenced, they'll send them back to me as files holding sequences of nucleotides. I can search for sequences similar or identical to my sequences using the NCBI website's BLAST function, which will compare my sequences to a large database.
DNA sequences are considered a more reliable means of identifying species for many reasons. For one thing, choosing morphological characters that are a useful diagnostic for defining species (especially fungal species) can be really hard. It's difficult to know whether all members of a species have a trait, or if a trait is shared with other species, so the trait may not be a defining characteristic. There also can be subjectivity in how different people measure the trait.
In contrast, a DNA sequence can act as a unique "barcode" hundreds of characters long and is read reliably through sequencing. When the right sequence of DNA is selected from a region in the genome, its order of nucleotide bases (A, T, G, C) should vary between species, but not within species. With each base acting as a character, DNA barcoding can also give us information about how closely related species are to each other, because more closely related species should have more similar barcoding regions. (However, to get a more accurate family tree, multiple regions of the genome must be sequenced and compared.) The common barcoding region for fungi (also frequently used for plants) is the ITS region, or the Internal Transcribed Spacer region. This stretch of DNA sits between genes which code for different parts of the ribosome.
So, I'll be using DNA barcoding to identify the species of host and parasite in the lobster mushroom association. I can also build family trees, or phylogenies, to determine whether specialization may have evolved in the parasite--if different branches of the parasite phylogeny are found on different hosts, this could imply that they have evolved a preference for certain hosts.
To obtain my DNA from dried samples, I'll use an extraction procedure that lyses (breaks open) cells so that the DNA is exposed. Some extraction procedures include steps for filtering out other unwanted cellular components like proteins that may degrade DNA. Next, I'll need to copy the extracted DNA using polymerase chain reaction (PCR). PCR uses an enzyme called DNA polymerase, which copies the DNA in our bodies during mitosis and meiosis. PCR has many cycles of different temperates which break double stranded DNA apart into single strands (high temp), allow short pieces of DNA complimentary to the region being copied called primers to anneal to the single strands (lower temp), and activate the DNA polymerase (medium temp). These three steps occur over and over again, doubling the amount of DNA in each cycle.
Once the DNA has been copied through PCR, it will be cleaned and prepared for sequencing. My lab sends out our samples to the sequencing core at our university. They have machines which perform Sanger sequencing, a process that uses fluorescently tagged nucleotide bases to determine the sequence of bases in my sample. Once the samples have been sequenced, they'll send them back to me as files holding sequences of nucleotides. I can search for sequences similar or identical to my sequences using the NCBI website's BLAST function, which will compare my sequences to a large database.
Comments
Post a Comment