Single gene sequencing
Single Gene Sequencing
We also want to know the exact sequence of some genes used to barcode individuals, like a barcode on a book you might buy at a bookstore!
The capillary sequencer can’t see the DNA sequence. We incorporate fluorescently-dyed nucleotides into a sequence of interest. This happens in several steps.
First, the DNA is copied many times.
Second, we begin to incorporate a fluorescently dyed nucleotide which stops the copying of that piece. We end up with some pieces that end in a T, some in A, some in C and some in G.
We, then, pass the PCR products through the capillary sequencer and the laser in the sequencer causes the different dyes to fluoresce.
A software program then reads the bases as the pieces pass by the laser and slowly a large basepair sequence is constructed.
We then have to polish each sequence using another program. Sometimes, some basepairs are hard to read so we have to look at the raw data.
In the end, we obtain a 500 or so basepair sequence. We, then, use another program to compare the order of nucleotides in one individual with another individual. We can then calculate the proportion of differences between the sequence of two or many individuals.
The variation at single genes is much lower than at microsatellites as often these genes code for something important. Thus, they can’t vary too much otherwise the function they provide may not work.
Stacy and Sarah were using a single gene in the mitochondrial genome of Gracilaria vermiculophylla to help trace the invasive history. They also applied restriction enzymes to this gene which meant they didn’t have to sequence and clean up over 2000 individual sequences!!
Alyssa is using a nuclear single gene to help her construct her seaweed phylogeny of red and brown seaweeds from San Diego, Antarctica and Fiji!
We also want to know the exact sequence of some genes used to barcode individuals, like a barcode on a book you might buy at a bookstore!
The capillary sequencer can’t see the DNA sequence. We incorporate fluorescently-dyed nucleotides into a sequence of interest. This happens in several steps.
First, the DNA is copied many times.
Second, we begin to incorporate a fluorescently dyed nucleotide which stops the copying of that piece. We end up with some pieces that end in a T, some in A, some in C and some in G.
We, then, pass the PCR products through the capillary sequencer and the laser in the sequencer causes the different dyes to fluoresce.
A software program then reads the bases as the pieces pass by the laser and slowly a large basepair sequence is constructed.
We then have to polish each sequence using another program. Sometimes, some basepairs are hard to read so we have to look at the raw data.
In the end, we obtain a 500 or so basepair sequence. We, then, use another program to compare the order of nucleotides in one individual with another individual. We can then calculate the proportion of differences between the sequence of two or many individuals.
The variation at single genes is much lower than at microsatellites as often these genes code for something important. Thus, they can’t vary too much otherwise the function they provide may not work.
Stacy and Sarah were using a single gene in the mitochondrial genome of Gracilaria vermiculophylla to help trace the invasive history. They also applied restriction enzymes to this gene which meant they didn’t have to sequence and clean up over 2000 individual sequences!!
Alyssa is using a nuclear single gene to help her construct her seaweed phylogeny of red and brown seaweeds from San Diego, Antarctica and Fiji!