Theme 4 Module 3

• Cells have an innate copying mechanism that allows for the semiconservative replication of DNA with each mitotic cell division
• The mechanism of replication is similar in prokaryotes and eukaryotes, and the process of DNA replication requires essential protein replication machinery that are needed for the synthesis of daughter DNA strands from parental strands in all cells
• Polymerase chain reaction (PCR)


  Technique developed by Kary Mullis in 1985 that allows scientists to copy (or amplify) millions of copies of DNA from very small starting samples
• PCR


  • It has revolutionized the world of cellular and molecular biology
  • It has shed light on diagnosis of genetic defects, detection of viral DNA in cells, producing large amounts of DNA from fossils containing trace amounts of DNA, and even being able to link specific individuals to DNA samples during forensic investigations
• Setting up a PCR

  1. Place a sample of DNA into a tube containing a buffered solution with essential ions and salts, along with a pair of short single-stranded DNA primers

  2. Add free deoxyribonucleotides (dNTPs)

  3. Add a heat-tolerant DNA polymerase (e.g. Taq polymerase)

  4. Place the tubes in a thermocycler machine that goes through various phases of heating and cooling to facilitate the DNA replication process over various repeated cycles
• Stages of a PCR cycle

  1. Denaturation

  2. Annealing

  3. Extension
• Each complete PCR cycle results in two double stranded helices containing the desired target sequence portion of the original template DNA
• With each successive PCR cycle


  The number of replicated DNA molecules with the same sequence as the parent template duplex is doubled
• Gel electrophoresis


  A technique that can be used to separate DNA fragments, RNA, and proteins based on their rate of movement through an agarose gel in an electric field
• DNA gel electrophoresis


  1. Load DNA samples into wells of a porous gel

  2. Apply an electrical field along the length of the gel

  3. Smaller molecules travel faster through the gel pores than larger molecules

  4. Visualize the separated DNA fragments using fluorescent dyes and UV light
• DNA sequencing was developed in 1975 by Frederick Sanger to determine the sequence of a DNA molecule
• Shotgun sequencing


  An approach to large-scale sequencing projects that involves breaking the entire genome into different sized pieces, randomly sequencing the DNA in each fragment, identifying regions of overlap between the fragments, and assembling the long, continuous sequence of nucleotides
• The first bacterial whole genome to be sequenced was that of Haemophilus influenza in 1995, followed by the first multicellular organism genome of C. elegans in 1998 and Drosophila in 1999, paving the way for the sequencing of the whole human genome in 2000
• Sanger sequencing (dideoxy chain-termination method)


  A DNA sequencing technique that utilizes modified deoxyribonucleotides (ddNTPs) that lack the 3'-OH group, causing DNA synthesis to terminate when incorporated
• xy chain-termination method


  Also known as dideoxy sequencing
• DNA sequencing


  1. DNA to be sequenced serves as a template for DNA synthesis

  2. Key components required: denatured, single-stranded template DNA

  3. Short single-stranded DNA primers that bind to template DNA

  4. Free deoxyribonucleotides (dNTPs)

  5. DNA polymerase
• Dideoxynucleotides (ddNTPs)


  Modified deoxyribonucleotides missing the 3'-OH group, preventing further elongation of DNA strand
• Dideoxy sequencing


  1. Synthesis of daughter strands begins at 3' end of primer and continues until a ddNTP is inserted

  2. Insertion of ddNTPs and dNTPs is a random process

  3. Generates many fragments of different sizes terminating at every possible nucleotide
• Fluorescent dyes


  Used to label each of the four ddNTPs, allowing identification of chain terminators
• Gel electrophoresis


  1. Labelled strands are separated by length, with shorter strands moving faster

  2. Laser excites fluorescent dyes, detector records fluorescence and matches to ddNTP

  3. Spectrogram trace generated, with peaks corresponding to nucleotide sequence
• Sanger dideoxy chain-termination method can only determine sequence of DNA fragments up to several hundred nucleotides
• Shotgun sequencing


  Sequences random DNA fragments, identifies regions of overlap, assembles into long continuous sequences
• Contigs


  Overlapping DNA segments assembled into a consensus region of DNA
• Genome annotation


  Identifying reading frames, protein-coding regions, regulatory sequences, and other sequence motifs
• Eukaryotic genomes contain a large proportion of non-coding and repeated sequences, contributing to diversity