Research Methods 2.7 DNA Sequencing

DNA sequencing

determines the exact order of nucleotide bases

chain termination methods

1. single stranded template of DNA uses

2. 4 test tubes with DNA

3. DNA syntehesi

4. Electrophoresis

4 test tubes with DNA

sequencing primer that will start DNA synthesis, must be specifically designed for each sequence, plasmid sequence is known and primer sequences can be designed. also DNA polymerase required along with DNTPs that are present in excesse and labeled with fluorescent tags. finally, ddNTPs which lack 3’OH needed for elongation

DNA synthsis

allow DNA synthesis to proceed, almost all DNTPs incorporated, but addition of ddNTPs makes synthesis stop because the next phosphodiester bond cant be formed. sequencing reactions are set up with both templates DNA and dNTPs in excess. DNA synthesis happens many times within the same tube on different template strands. synthesis incorporated ddNTPs to stop synthesis

gel electrophoresis

polyacrylamide gel electrophoresis separates by size, shorter at bottom, largest at top (OLD form)

evolution of automated sequencing

sequences of longer DNA

modifications

use fluorescent tag and capillary electrophoresis, cycle sequencing also uses less starting material because several DNA fragments could be made from the same template.

dye terminator sequencing

4 different dyes used allowing sequencing to be done as a single reaction, plasmid is mixed with a single primer which binds in the aneealing step and extension progresses with dNTPs until a random fluorescently labeled ddNTP is incorporated into the growing strand

capillary electrophoresis

DNA separated in a capillary, electrical current separates by size, exits in order of smallest to largest. as DNA fragments exit, dyes on ddNTPs are excited by a laser and emitted light is detected.

chromatogram

bases represented by a sequence of colored peaks and peak height indicates intensity of flurorescent signal

cycle sequencing

successive rounds of denaturation, annealing, and extension in thermal cycler results in linear amplification of extension products. an advantage is that less template is required because the reactions are in the same tube. also, by knowing the size of the DNA strand and the last base that terminated the sequence, identification of the sequence can be done

primers

single oligonucleotide per reaction tube, 4 primers are used for full and deep coverage because the sequence is longer then 500-800bp.

primers used outside of the MCS

• pJET seq f (blue)

• pJET seq r (yellow)

primers used to anneal to GAPC gene

• GAP seq f (red)

• GAP seq r (green)