DNA SEQUENCING

• DNA comprises some three billion base pairings (3 gigabases of DNA) in the human genome
• Genome


  The entire DNA sequence of an organism
• Only a small amount of DNA actually consists of coding DNA – a mere 1.5% of human DNA codes for polypeptides, the rest junk DNA (also called non-coding DNA)
• Sanger sequencing


  The most common method of sequencing used nowadays, introduced by Frederick Sanger in 1975, also known as the chain termination method
• Sanger sequencing


  1. Template DNA is copied repeatedly, with the copies terminating at different points

  2. Copies are arranged into order of size to determine the sequence
• Sanger sequencing


  1. Reaction is initiated at either end of the target length of DNA

  2. Two strands separate

  3. Primer binds to template

  4. DNA polymerase builds complementary strand

  5. Fluorescently-marked nucleotide (dideoxynucleoside triphosphate) is placed in strand

  6. Fragments are collected and separated using electrophoresis
• Dideoxynucleosides lack a hydroxyl group (OH) in their structure, which prevents DNA polymerase from binding further bases to the sequence after the dideoxynucleoside has joined
• Polymerase chain reaction (PCR)


  A process used to mass produce the target DNA fragments required for Sanger sequencing
• Polymerase chain reaction (PCR)


  1. Double-stranded DNA sample is heated to separate strands

  2. Short DNA primers complementary to the ends of the target DNA anneal to the single strands

  3. DNA polymerase (taq polymerase) binds free nucleotides to build parallel strands

  4. Process is repeated, exponentially increasing the number of copies
• Taq polymerase


  • A thermophilic DNA polymerase that can function at high temperatures without being denatured
• Sanger sequencing comprises PCR, the sequencing reaction and electrophoresis
• Sanger sequencing


  1. Target DNA is replicated using PCR

  2. Sequencing reaction takes place with primers, nucleotides and dideoxynucleosides

  3. Resultant DNA fragments of different lengths are separated by electrophoresis

  4. Laser reads the fluorescent markers to determine the DNA sequence
• After 25 cycles of PCR, the single strand at the beginning of the process has been replicated to produce just over 33 million lengths of DNA
• DNA probe


  A short, single-stranded piece of DNA around 50 bases long which can be used to test for the presence of a particular sequence of DNA
• DNA probes


  • They can be labelled with radioactive, fluorescent or antigen markers
  • They bind to complementary DNA sequences through annealing
• Southern blotting is used to further analyse DNA fragments separated by electrophoresis
• Southern blotting


  1. A nylon sheet is placed over the gel plate and DNA fragments are transferred to the sheet by capillarity

  2. Radioactive or fluorescent markers are used to visualise the DNA fragments on the sheet
• Genome


  The entire DNA sequence of an organism, containing all the genetic information necessary for the development and function of that organism. Includes both genes and non-coding DNA.
• Genotype


  The specific alleles (versions) of genes that an organism has for a particular trait. Refers to the genetic makeup of an organism with respect to a specific trait.
• Chain termination


  The method used in Sanger sequencing to determine the order of nucleotides in a DNA strand, involving the use of a mixture of regular nucleotides and modified nucleotides called dideoxynucleotides (ddNTPs), which terminate the chain when incorporated into the growing DNA strand
• Chain initiation


  The first step in DNA replication, where a DNA polymerase enzyme adds a nucleotide (a building block of DNA) to the beginning of a new DNA strand