biotech - dna sequencing

Human Genome Project

• Genome: an organism’s complete set of DNA.

• Human genome contains ~3 billion base pairs.

• The Human Genome Project (1998–2003) was an international effort to sequence and map all human genes.

Competing Institutions

• Celera Genomics (private company headed by Craig Venter)

• sequencing approach: shotgun

• description of method: Randomly breaking up DNA sequences into lots of small pieces, sequencing the fragments and reassembling them by looking for regions of overlap

• Public research institutions worldwide (USA, UK, France, Germany, Japan, China)

• Small scale shotgun

• Same as shotgun except that markers are used at regular intervals in the genome to make it easier to reassemble the sequence

Fredrick Sanger (1975)

• Developed dideoxy termination sequencing method

• 1980 received Nobel Prize in Chemistry for sequencing method

• 1958 received Nobel Prize in Chemistry for his work on the structure of insulin

Dideoxyribonucleotides (ddNTP)

Di = two, Deoxy = removed oxygen  Dideoxy NTPs have oxygens missing at both the 2’ and 3’ position

Polymerization Reaction

 3’OH is needed to react with the phosphate group on the 5’ end of the next nucleotide to form a phosphodiester bond

ddNTP = dideoxynucleotide triphosphate

• A modified DNA nucleotide that lacks a 3′-OH group

• When added during DNA replication, it stops the chain

• ddATP specfic type of ddNTP

What do you think will happen if you allow DNA replication to occur in the presence of both deoxyATP and dideoxyATP?

The newly synthesized strands of DNA have different lengths depending on when a ddNTP was incorporated into the strand

Each tube is designed to stop DNA at one specific base:

• Tube A → stops at A

• Tube G → stops at G

• Tube T → stops at T

• Tube C → stops at C

• A small amount of one special “stop” nucleotide (ddATP, ddGTP, etc.) is added

• dNTP (normal) → DNA keeps growing

• ddNTP (special) → DNA STOPS immediately

• Mostly normal nucleotides (so DNA usually keeps growing)

• A small amount of ddNTP (so sometimes it stops)

👉 This creates many DNA fragments of different lengths, each ending at that base.

Sanger Sequencing Setup

• Each reaction vessel only has ONE of the 4 possible ddNTPs

• the strands of different lengths will be separated using gel electrophoresis onto 4 lanes, one for each ddNTP reaction

Modern sequencing

• Same principle as Sanger sequencing, but simplified.

• Four ddNTPs are labeled with different fluorescent dyes in one tube.

• All fragments are run together in a single gel lane.

• Fragments are still separate by size but show up as coloured bands

• Colours have different wavelengths which can be read by a computer

• Computer translates the colours into the order of the nucleotides