BIO2015: Lecture 4

DNA sequencing

·      Use provides order of the nucleotides in given DNA

·      2 main methods:

·      1) Sanger dideoxynucleotide chain termination method (commonly used method):

·      a) manual method

·      b) automated method

·      2) chemical cleavage method (maxam and gilbert method)- not used nowadays

·      sanger dideoxynucleotide chain termination method- This method relies on use of deoxyribonucleoside triphosphates, derivatives of the normal deoxyribonucleoside triphosphates that lack the 3’ hydroxyl group

·      purified DNA synthesized in vitro in mixture that contains:

·      single stranded molecules of the DNA to be sequenced

·      enzyme DNA polymerase

·      short primer DNA to enable polymerase to start DNA synthesis

·      the 4 deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP: A, C, G, T)

·      if dideoxyribonucleotide analog of one of these nucleotides is in the nucleotide mixture- can become incorporated into growing DNA chain

·      this chain now lacks 3’ OH addition of next nucleotide is blocked, and DNA terminates at this point

·      so for e.g. if the analog is of dATP, it competes with normal dATP so that analog ddATP is occasionally incorporated at random into growing chain

·      reaction mixture will then produce set of DNAs of different lengths complementary to template DNA being sequenced and terminating at each of the different A’s

·      exact lengths of the DNA synthesis products can then be used to determine position of each A in growing chain

·      to determine complete sequence of DNA fragment- double stranded DNA first separated into single strands and one of the strands is used as template for sequencing

·      4 different chain terminating analogs are used in 4 separate reactions on copies of the same single-stranded DNA template

·      each reaction produces set of DNA copies that terminate at different points in the sequence

·      products of these 4 reactions are separated using gele electrophoresis in 4 parallel lanes of a polyacrylamide gel

·      newly synthesized fragments are detected by a label (either radioactive or fluorescent) that has been incorporated either into primer or into one of the deoxyribonucleoside triphosphates used to extend the DNA chain.

·      In each lane of the polyacrylamide gel- bands represent fragments that have terminated at a given nucleotide but at different positions in the DNA

Labelling methods

·      Labeling the nucleotides:

·      S³⁵ dNTP or P³² or fluorescent labelled dNTP

·      Labeling the primers:

·      S³⁵ dNTP or P³² or fluorescent labelled dNTP

·      Radioactive labeling= manual sequencing

·      Fluorescent labeling= automated sequencing

·      Typical read lengths of around 800 bases

Improvements to sanger

·      Sequencing was developed 30 years ago why have genomes only recently been sequenced?

·      15-20 years ago-…