Lec 23 & 24

• DNA Sequencing and the Human Genome

  1. DNA sequencing

  2. Bioinformatics in genome analysis
• Molecular techniques to be discussed

  • Animal cloning
  • Molecular cloning
  • DNA sequencing
  • Cytogenetics (Karyotyping)
  • Microarray technology
  • PCR
  • Genomics Application
• History of genetic analysis

  1. Mendelian research

  2. Genetic disorder inheritance (Haemophilia)
• DNA


  Carrier of genetic information<|>Source of genetic information found in chromosomes<|>Dictates amino acid sequence in proteins<|>Information in chromosomes is then passed from parent to offsprings
• Transforming principle


  Substance that could be transferred from non living cells to living cells, causing the living cell to show characteristics of the non living cell
• Biotechnology Applications in Veterinary Medicine & Genetics

  • Artificial insemination
  • Multiple ovulation and embryo transfer
  • In vitro maturation and in vitro fertilization
  • Transgenic animals
  • Animal cloning
  • Vaccines or antibiotics development
• Transgenic animals


  Eukaryotes also subject to DNA transformation<|>DNA via cytological studies had shown resides in the chromosomes, must also be the hereditary material of eukaryotes<|>Genetically altered animals are called transgenic
• Animal Cloning


  1. Production of genetically identical animals

  2. Naturally occurring in identical twins, natural splitting of an embryo soon after fertilization

  3. Embryo splitting made possible in the lab

  4. Each portion of the split embryo inserted into a foster mother, brought to term in a usual way

  5. Cloning by nuclear transfer, involves obtaining unfertilized ova from a recipient female, removing nucleus from each ovum and replacing it with a nucleus (or more commonly a whole cell) from a tissue or cultured cells
• Manipulation of RNA genome in vaccines development
• Manipulation of RNA polymerase/RNA transcription in antibiotics development
• Techniques of Molecular Genetics

  • Recombinant DNA Technology
  • Genetic engineering
  • Locating, isolating, altering and studying DNA segments
  • Biotechnology - Using recombinant DNA technology to develop new biological products
• Recombinant DNA technology


  Involves isolation, recombine and amplifying genes<|>One molecule composed of two distinct DNA sources<|>Requires the use of restriction endonucleases/enzymes to make double-stranded cuts in DNA<|>Bacterial source name of enzymes is an abbreviation of bacterial source<|>Usually recognizes 4-6 palindromic sequences
• Cutting and Joining DNA Fragments

  1. Restriction enzymes: recognizing and cutting DNA at specific nucleotide sequences

  2. Palindromic sequences

  3. Immune system of bacteria

  4. Type II restriction enzyme: most useful enzyme

  5. By adding methyl groups to the recognition sequence to protect itself from being digested by its own enzyme in bacteria

  6. Cohesive ends: fragments with short, single-stranded overhanging ends

  7. Blunt ends: even-length ends from both single strands
• Viewing DNA Fragments

  1. Gel electrophoresis: Porous gel made of agarose or polyacrylamide, Sample DNA mixed with loading dye, Negatively-charged DNA runs toward positive pole when electrical current passes through the gel, Separates fragments based on size

  2. Ladder or marker contains fragments of known sizes to aid in determination of sample fragment size

  3. Expose gel to dye: Methylene blue - light box, Ethidium bromide - UV light

  4. Locating DNA fragments with Southern blotting and probes: Probe is DNA or RNA with a base sequence complementary to a sequence in the gene of interest, Usually labeled for easy detection: Radioactive P32, Fluorescent tag
• Type II restriction enzyme


  Most useful enzyme
• Type II restriction enzyme


  Adds methyl groups to recognition sequence to protect itself from being digested by its own enzyme in bacteria
• Cohesive ends


  Fragments with short, single-stranded overhanging ends
• Blunt ends


  Even-length ends from both single strands
• VPP3140 Veterinary Genetics
• Gel electrophoresis


  • Porous gel made of agarose or polyacrylamide
  • Sample DNA mixed with loading dye that allows for visualization and increases density
  • Negatively-charged DNA runs toward positive pole when electrical current passes through the gel
  • Separates fragments based on size
  • Smaller fragments migrate the furthest - bottom of the gel
  • Ladder or marker contains fragments of known sizes to aid in determination of sample fragment size
• Viewing DNA Fragments

  1. Expose gel to dye

  2. Methylene blue - light box

  3. Ethidium bromide - UV light
• Locating DNA fragments with Southern blotting and probes

  1. Probe: DNA or RNA with a base sequence complementary to a sequence in the gene of interest

  2. Is usually labeled for easy detection

  3. Radioactive P32

  4. Fluorescent tag
• Amplifying DNA fragments with PCR

  1. Taq polymerase: stable DNA polymerase at high temperature

  2. Researcher designs specific oligonucleotide primers that serve as the ends of the amplified fragment

  3. Very similar to replication
• Central dogma of molecular biology
• What is a genome?
• DNA sequencing


  The process of determining the sequence of nucleotide bases (A, T, C and G) in a piece of DNA
• Ingredients for DNA sequencing

  • DNA template
  • DNA polymerase
  • Primers
  • Four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
• Sanger's dideoxy-sequencing method

  Dideoxyribonucleoside triphosphate (ddNTP) lacks a 3'-oh group, which terminates DNA synthesis
• Sanger Method - Capillary Electrophoresis
• Trends of DNA sequencing
• High throughput sequencing in the lab
• High throughput sequencing data & analysis
• Bioinformatics in genome analysis
• Veterinary Genomics


  An area within genetics involving the sequencing and analysis of an organism's genome - and the implications of genomics for veterinary medicine and human health
• DNA Fingerprinting (DNA Profiling)


  Technique used to distinguish between individuals of the same species using DNA samples<|>Microsatellites: variable number of copies of repeat sequences possessed by many organisms, which can be amplified by PCR<|>Combined with RFLP analysis to form more thorough fingerprint
• DNA Fingerprinting Process

  1. Cells broken down to release DNA

  2. Amplification by PCR (small amount DNA)

  3. Pattern of fragment distribution analysed

  4. DNA fragments into agarose gel

  5. Restriction enzymes
• Forward genetics


  • Begins with a phenotype to a gene that encodes the phenotype
• Reverse genetics


  • Begins with a gene of unknown function, first inducing mutations and then checking the effect of the mutation on the phenotype
  • Site-directed mutagenesis: creating mutation in particular DNA sequences, and then studying the effects of these mutation on the organisms
  • Silencing genes with RNAi: Using RNAi for the treatment of human disease: lowering ApoB with RNAi
• Model Genetic Organism


  The mouse, Mus musculus
• Applications of Genetic Engineering

  • Pharmaceuticals
  • Human insulin
  • Specialized bacteria
  • Agricultural products
  • Oligo nucleotide drugs
  • Genetic testing
  • Gene therapy: Direct transfer of genes into humans to treat disease
  • Bioremediation: Bacteria genetically engineered to break down toxic chemicals
  • Agriculture: Viral/pesticide resistance; increase nutritional value