How to Sequence DNA Using the Sanger Method
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How to Sequence DNA Using the Sanger Method

After reading this you should understand all aspects of Sanger Sequencing. You will understand what components are used and the method used to sequence DNA. You will also understand what a ddNTP is and how to read the polyacrylamide gel. Finally you will understand how Sanger Sequencing is so important for genetics.

Sanger Sequencing is a method used to figure out the sequence of a single-strand of DNA. This process done in the lab and requires the use of: one type of primer, ddNTP’s, dNTP’s, buffer solution, polymerase and a DNA strand. The primer is a small know sequence of DNA that starts the Replication. Next we have the ddNTP’s which are like nucleotides but they stop replication because since they are lacking the OH that is bound with a phosphate. Then when have the dNTP’s, which are nucleotides that allow for normal replication. We also have a buffer solution and a polymerase to ensure replication proceeds.

The process begins by taking a test tube and inserting all of the above ingredients into it. One kind of dNTP needs to be marked radioactively, but all four dNTP’s need to be in the tube unmarked as well. There should be four tubes: one with radioactively marked ATP, one with radioactively marked GTP, one with radioactively marked TTP, and one with radioactively marked CTP. You allow the solution to sit for a couple minutes and then end up with a solution of DNA strands of varying lengths. These varying lengths of DNA are due to the fact that there are ddNTP’s stopping replication at different spots on the DNA strand.

Then you run the four different solutions on a polyacrylamide gel. When this is done the ddATP column will have all lengths of DNA that stop at an A nucleotide and the ddGTP will have all lengths of DNA that stop at a G nucleotide and the same thing will be true for the ddTTP and ddCTP columns. This means that the polyacrylamide gel now displays every possible length of DNA. Now the gel just needs to be read in order.

Starting from the top of the gel you read down in order from the line that shows up first. If the column contained ddATP and there is a line there then you read A. Once you have read the entire gel you have to find its DNA compliment because you replicated it to begin with. For example if you read “ATGCTA” the actual strand will be “TACGAT” because A codes for T, G codes for C, and vice versa. Now you have completed sequencing a strand of DNA.

Sanger Sequencing was among the first sequencing methods and is still used throughout labs today. Sanger Sequencing started a new era of genetics and is responsible for things like the human genome project and the basis behind polymerase chain reaction (PCR).

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