The principles of DNA sequencing

Question 1

Process of DNA sequencing

Answer 1

1.Into tube DNA for sequencing, primer, DNA polymerase, excess of normal nucleotides and terminator bases
2.Mixture placed in a thermal cycler
3.Heated to 96 degrees so double-stranded DNA separates into single strands
4.Cooled down to 50 degrees so primers anneal to DNA strand
5.At 60 degrees DNA polymerase starts to build up new DNA strands by adding nucleotides with complementary base to single-stranded DNA template
6.Each time terminator base is incorporated instead of normal, synthesis is terminated. These are present in lower amounts and added at random, resulting in many DNA fragments of different lengths. After a few cycles, all possible chains have been produced.
7.Capillary sequencing
8.Fluorescent markers on terminator bases are used to identify final base on each fragment. Lasers detect different colours and thus the order of the sequence.
9.Order of bases in capillary tubes shows sequence of new, complementary DNA. This is used to build up sequence of original DNA strand.
10.Fed into computer that resembles genomes by comparing all fragments and finding areas of overlap between them.
11.Once genome assembles, scientists want to identify the genes or parts of the genome that code for specific characteristics.