DNA Sequencing and Dideoxynucleotide Method

DNA Sequencing

• DNA sequencing is the process of determining the order of nucleotides in a DNA molecule.
• A deep understanding of a DNA molecule is based on knowing its nucleotide sequence.
• Knowing the sequence allows deduction of the function of genes by comparing them to known genes.
• Sequence information is crucial in detecting single-base mutations.

Dideoxynucleotide Method

• The dideoxynucleotide method, developed by F. Sanger, is the most common DNA sequencing procedure.
• Sanger received Nobel Prizes for inventing sequencing techniques.
• A dideoxynucleotide lacks a hydroxyl group (OH) on both the 2' and 3' carbons of the sugar moiety.

DNA Replication and Sequencing

• During DNA replication, natural nucleoside triphosphates are incorporated into the growing chain based on complementary base pairing.
• These are connected with a 5' to 3' phosphodiester bond.
• During sequencing, dideoxynucleotides are added. If a dideoxynucleotide is added, DNA synthesis stops. This is because a regular phosphodiester bond cannot form.

DNA Sequencing Procedure

• The method begins by annealing a synthetic oligonucleotide primer to a DNA template.
• Primers are typically 17-24 bases long.
• The primer should base pair to a highly specific region.
• A primer provides a 3'-hydroxyl group for initiating DNA synthesis
• The DNA sample is split into four separate reaction tubes.
• Each tube contains four deoxyribonucleotides (dATP, dCTP, dGTP, and dTTP), one radiolabeled dideoxynucleotide (ddATP, ddCTP, ddGTP, or ddTTP), and DNA polymerase.
• The dideoxynucleotide concentration is carefully controlled to incorporate at every possible site, not just the first place.

Sequencing Data Interpretation

• Formamide is added to stop DNA synthesis and prevent base pairing.
• Reaction products in each tube are separated using polyacrylamide gel electrophoresis.
• Separated DNA fragments of different sizes are visualized on an autoradiograph.
• The sequence is determined by the order of bands on the autoradiograph.
• Band order corresponds to the order of nucleotides in the original sequence.

Automated DNA Sequencing

• Modern sequencing instruments use fluorescent dyes.
• Dideoxynucleotides are fluorescently labeled.
• Four different fluorescent dyes, one for each nucleotide.
• Separated fragments are detected by a laser scan.
• This system is known as '4-color, 1-lane' detection.

Automated Results

• Emission data is recorded and stored in a computer.
• Computer converts the data into a readable nucleotide sequence.
• Modern systems can read about 500 bases per run and, under optimal conditions, resolve approximately 20,000 bases per hour.