- Method of DNA Sequencing – Sanger Sequencing
- Interpret a Sanger Sequence
Learn.
Sanger Sequencing allows DNA to be replicated then sequenced to determine the order of nucleotides in a DNA sequence.
The DNA which is being sequenced is first amplified – making many copies. Then the DNA is denatured through heating it up.
By incorporating a small amount of dideoxyATP (ddATP) after annealing, the polymerase will incorporate the ddATP into it in substitute for Adenine and terminate the reaction. Since the ddATP contains a fluorescent tag, this will occur at all sites containing adenine. This is repeated for ddTTP, ddGTP and ddCTP. In all instances dNTPs and primers are added to the reaction vessel.
Through exploiting DNA polymerases requirement for a 3’OH to create a complimentary chain, sanger sequencing constructs fragments with varying lengths since they are terminated by the ddNTPs.
Following this, all the fragments of differing lengths can be separated by gel electrophoresis. The negative phosphate of the sugar phosphate backbone in the DNA fragments can be exploited to separate the fragments. By placing them in a special polyacrylamide gel and applying a voltage, the gels move from the negative to the positive. However, the smaller fragments move faster.
When determining the sequence of the DNA. You read the gel electrophoresis from the positive end to the negative end of the gel electrophoresis. As a result you read the shortest fragment to longest fragment. Then to determine the sequence of the template strand you read the complimentary bases of the sequence you derived.
Memorize.
Master.
Question 1.
Compare and contrast polymerase chain reactions and sanger sequencing.
Question 2.
Outline the implications of contaminants in sanger sequencing.
Question 3.
Explain whether ddNTPs could replace dNTPs in sanger sequencing.
Question 4.
A student incorrectly read a sanger sequence. They said the sequence was:
ATATCGTACGCA
They read this from the negative end to the positive end of the gel electrophoresis and inverted the bases. Explain what the sequence should actually be.
