the human genome project

sanger sequencing

• DNA clones to be sequenced are generated by standard PCR reaction 

• The clones are then subject to a polymerase-mediated synthesis step

  • Critical is the random termination of extension at each nucleotide position 

• reaction mixture contains DNA template, primer, DNA polymerase, ddNTPs and dNTPs

• The random termination results in DNA fragments of varying sizes which can be analysed to determine nucleotide sequence 

ddNTP

• ddNTP have a hydrogen replaced from hydroxyl in dNTP 

  • The hydroxyl is important for addition of next nucleotide 

  • Thus next nucleotide is not able to be added 

• dNTPs are added in excess over ddNTPs in reaction mixture

• Termination Is random 

  • But will have termination at every single position 

  • Provided there are enough clones 

• ddNTPs are labelled but dNTPs are not

  • with fluorophores

reading DNA

• Need a primer 

• Run on a polyacrylamide gel 

• Shortest DNA fragments run faster 

  • Will be at the bottom 

fluorescence detection systems

• Fluorescence detection systems are used to image the fluorescence producing an electropherogram 

  • Peaks is due to number of DNA strands with that sequence 

• Capillary electrophoresis allows the standard gel electrophoresis step to be bypassed 

• Further technical upgrades meant that 384 samples could be run in parallel 

sequencing the human genome for the first time

• Sanger sequencing yields only up to a maximum of 1000 nucleotides per reaction 

  • Challenge for large scale projects 

• The sequencing strategy was shaped by 3 key limitations of sanger sequencing 

  • The necessity to have a clone of the DNA template (so that the levels of fluorescence emitted is able to be detected) 

  • The requirement that at least some sequence information is known beforehand (so that primers can bind to the template) 

  • The short sequencing read length 

• Due to using a vector, the primer can be designed to be complimentary to the vector 

process

• Fragment a chromosome 

• Order them 

• Fragment those fragments 

• Sequence 

• Reorder fragments 

problem = fragmentation happens randomly

ordering larger fragments

• Chromosomal DNA was initially fragmented into large pieces and cloned into vectors known as YACs (yeast artificial chromosomes) 

• The clones were then mapped in terms of their original chromosomal location by 2 major techniques 

  • FISH-type experiments 

    • Label specific sequences 

    • Can be detected on both chromosome and fragments 

    • Can thus be reordered 

  • PCR-based screening for STSs 

    • Bits have already been sequenced 

    • Called sequence tagged sites 

    • Use sequence to design a complimentary primer 

    • One that is positive thus has the same sequence and comes from the same location 

• A series of overlapping clones whose chromosomal location had been mapped was thus generated and these were referred to as clone contigs 

  • No gaps 

reordering smaller fragments

• Do reaction so that fragmentation only happens at certain sites 

• Add restriction endonuclease at smaller concentrations  

• Random fragments 

• Overlap 

• And match overlapping ends (sequences of homology) 

whole genome shotgun sequencing

• Shotgun sequencing is used to determine the DNA sequence of an entire genome.

• The genome is broken into many small, random fragments.

• These fragments are cloned into vectors or prepared as sequencing libraries.

• Each fragment is sequenced individually.

• Sequencing produces many overlapping DNA reads.

• Computers compare overlaps between reads.

• Overlapping sequences are assembled into longer contigs.

• Contigs are ordered and oriented to reconstruct the genome.

• The randomness increases the chance that every region is sequenced.

• Shotgun sequencing is faster and more efficient than sequencing DNA in order.

capillary sequencing / automated Sanger sequencing

• Capillary sequencing is used to determine the nucleotide sequence of DNA fragments.

• DNA is first amplified (often by PCR).

• The sequencing reaction uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis.

• Each ddNTP is fluorescently labeled with a different color.

• DNA fragments of varying lengths are produced.

• The fragments are loaded into a thin capillary tube filled with polymer.

• An electric field separates fragments by size during electrophoresis.

• Shorter fragments move faster through the capillary.

• A laser detects fluorescence as fragments pass a detector.

• A computer reads the colors to determine the DNA sequence.