Biotechnology Tools and Techniques

• DNA Extraction

  • DNA extraction is the method of removing and collecting DNA from an organisms cell.
  • This method involves physical and chemical methods.
  • Due to this, DNA is able to be extracted, and potentially moved from one species to another.
  • These techniques are used in:
  • Forensics science
  • Paternity tests
  • Vaccines
  • Cloning
  • Many more
• DNA Extraction

  The method of DNA extraction includes the following:

  1. Obtaining cell/tissue samples
  2. Centrifuge (spins) cells at high speeds – separates nucleus from rest of the cell
  3. Add detergents to break down nuclear membrane – chromosomes are exposed
  4. DNA is isolated from histone proteins
• Gene Probes

  • Gene probes are single strand segments of DNA or RNA.
  • Are constructed in the laboratory.
  • Used to select particular readily located genes that are radioactive or have fluorescent markers.
  • The probes work by binding complementary to the genes of interest.
  • Probe will bind with gene and become hybridised.
  • Radioactive/fluorescent markers make it easier for gene to be located.
• Restriction Enzymes

  • Bacteria produce something called a restriction enzyme (RE) (endonucleases) which are able to cut DNA naturally by making an incision or cutting the sugar phosphate backbone.
  • This is used as a defensive method against viral DNA that invade the bacteria.
  • These enzymes were discovered in 1970 and used in genetic engineering to help remove and isolate genes.
  • Most RE’s recognise short nucleotide sequences: 4-8 nucleotides in length – referred to as restriction sites.
  • Enzyme EcoR1 was extracted from E. Coli which cuts in staggered fashion.
• Restriction Enzyme Examples

  Restriction Enzyme EcoR1

  • Enzyme cuts between the G and A bases.
  • Creates overlapping ends (sticky ends) – enables complementary binding between host DNA and donor DNA.
  • Enzyme DNA ligase is used to join the host and donor DNA.
  • Bacterial transformation - incorporation of DNA into bacteria
• Polymerase Chain Reaction (PCR)

  • Samples of DNA that scientists work with are usually quite small.
  • PCR was developed to help scientists create many copies of the same DNA prior to analysis of other applications.
  • DNA put through PCR process is said to have been amplified (multiplied).
• Polymerase Chain Reaction (PCR)

  • Target DNA templates – to be copied.
  • DNA primers – short sections of DNA which binds to either side of DNA templates to prevent DNA from reforming or Taq DNA polymerase from binding.
  • Free DNA nucleotides – used to assemble new DNA copies (building blocks).
  • Taq DNA polymerase – enzymes from bacteria that tolerate high temps. Binds DNA back together at the end.
  • Buffer solutions – provide suitable chemical environments.

  Note that sometimes mitochondrial DNA (mtDNA) is used and decreases need of PCR – due to many copies of mtDNA.
• The Three Stages of PCR

  Step 1: Denaturing

  • The template DNA is heated to about 95 degrees so that the hydrogen bonds holding the double helix together are broken and this exposes the bases

  Step 2: Annealing

  • The mixture is cooled to around 60 degrees and this enables the primers to bind to the complementary bases that are exposed on the template DNA
• three stages of PCR

  Step 3: Extension

  • The temperature is raised to approximately 72 degrees to enable the nucleotides to bind with the exposed DNA bases and the Taq DNA polymerase will then join them into a DNA strand. Each of the original strands has formed a new complementary strand at the end of the process
• The Three Stages of PCR

  Process is repeated. Many copies made in exponential fashion
• Gel Electrophoresis

  Two types of electrophoresis – gel and capillary.

  Electrophoresis is the process of separating molecules (e.g. DNA) based on size and charge.

  • Gel electrophoresis is the process of separating proteins or fragments of DNA according to size and charge (DNA is slightly negatively charged due to phosphate groups).
  • DNA samples are loaded into wells (indentations) at one end of the gel.
  • An electric current is applied to “push” the DNA through the gel.
  • Smaller fragments (less DNA base pairs) move further due to less resistance to agarose gel.
• Capillary Electrophoresis

  • Has multiple applications including DNA sequencing and DNA profiling.
  • Has mostly replaced gel electrophoresis due to being more automated and faster.
  • General principles of electrophoresis applies:
  • DNA fragments move from negative electrode to positive electrode.
  • Shorter DNA fragments move faster than longer ones.
  • Frederick Sanger developed a technique called chain termination method – allows DNA fragments to be fluorescently tagged.
  • Tagged on last nucleotide of DNA sequence. Different tags for each base.
• Capillary Electrophoresis

  • DNA fragments are loaded into capillaries containing a polymer and an electric field is applied.
  • Polyacrylamide gel is one such polymer.
  • Smaller DNA fragments travel faster and exit first.
  • DNA fragments are struck by a laser that excites the fluorescent dye of the last nucleotide.
  • As the fragment exits, a digital camera (detector) is used to capture the image.
  • Fluorescent images are converted into graphical representation called an electropherogram. These will differ depending on purpose of CE.
• DNA Sequencing

  • DNA sequencing involves techniques to determine the sequence of DNA bases within a gene/segment of DNA, or entire genome of organism.
  • First genome sequence of human was published in 2003, as a result of Human Genome Project (HGP).
  • Used in labs around the world to sequence genomes of different species.
  • Collecting and analysing this information continues to advance our understanding of genetics, gene control, and evolution.
  • This technology continues to develop, such as nanopore sequencing  in 2015
• DNA Sequencing

  DNA sequencing involves both Polymerase Chain Reaction (PCR) and capillary electrophoresis.

  1. DNA template is obtained.
  2. DNA template is mixed with Taq DNA polymerase, free floating DNA nucleotides, sequencing primers and terminator nucleotides (fluorescently tagged and terminate DNA synthesis).
  3. Reaction mixture runs through many PCR cycles – amplifies DNA.
  4. When fluorescently tagged terminator nucleotide is added by Taq polymerase, DNA synthesis is stopped.
• DNA Sequencing

  5. This results in array of DNA fragments of single base difference – due to termination at different places along DNA template.

  5. The array of newly synthesised DNA fragments undergoes capillary electrophoresis, identifying terminator nucleotide of each DNA fragment.

  6. DNA sequence electropherogram is produced.

  7. Sequence of DNA template is determined.
• DNA Profiling

  • Human cells contain roughly 3 billion base pairs
  • On average, about 99.9% of these base pairs are shared/the same across all individual humans (identical twins are the exception).
  • These variations are analysed in DNA profiling.
  • Even though humans share the same genetics, the 0.1% of DNA in humans is unique to those particular individuals.
  • 0.1% of DNA equals roughly 3 million base pairs.
  • This variation in DNA between individuals occurs in particular locations, called loci/locus
• Applications of DNA Profiling

  Innocence Project

  • Exonerating wrongfully convicted criminals

  Profile Archiving

  • Developing of state and national databases to record DNA profiles of citizens and use to match repeat offenders who may have evaded conviction in the past

  Forensic Science

  • Used as evidence to convict criminals to eliminate suspects, solve cold cases and reduce chance of wrongful conviction
• Introns in DNA Profiling

  • DNA Profiling relies on variation in non-coding introns.
  • Introns contain categories of repeating sequences.
  • Number of repeating sequences is unique to individual.
  • Two main categories of repeated sequencing:
  • Variable Number Tandem Repeats (VNTR).
  • Short Tandem Repeats (STR).
• Alleles vs Introns

  • An individual contains two sets of alleles (coding genes).
  • Individuals will also contain two sets of repeating introns (VNTRs and STRs).
  • Homozygous – same number of repeats for both chromosomes.
  • Heterozygous – different number of repeats for both chromosomes
• Variable Number Tandem Repeats (VNTR)

  • VNTR are repeated sequences of DNA bases.
  • Ranges between 10-80 bases in length.
  • Can repeat up to 30 times.
• Short Tandem Repeats (STR)

  • Has taken over VNTR in DNA profiling.
  • Are generally 2-6 bases in length and found at different loci in genome.
  • The variation in repeated introns results in differing DNA lengths.
  • Electrophoresis can be used to determine number of repeats.
• DNA profiling using gel electrophoresis

  1. Tested DNA is amplified using PCR.
  2. VNTR are cut out using restriction enzymes.
  3. DNA fragments are separated using gel electrophoresis.
  4. Southern blotting technique is used to view colourless DNA.
  5. DNA is transferred to a membrane.
  6. Addition of fluorescent probes that complementary bind to VNTR.
  7. Multiple fluorescent probes + multiple VNTR = unique pattern.
  8. DNA fragments and fluorescent probes are x-rayed.
• Interpreting DNA profiles – gel electrophoresis

  • Analysis of DNA profiles depends on type of DNA gathered:
  • Pure sample of perpetrators DNA
  • All DNA fragments need to align
  • Mixture of perpetrators and victims DNA
  • Not all DNA fragments match. Similar to paternity test
• Interpreting DNA profiles – gel electrophoresis

  • STRs are also used in gel electrophoresis to create DNA profiles.
  • Same process as VNTR is used except fluorescent probes used must be complementary to STR repeating unit.
  • Number of STR repeats is based on distance DNA fragments travel.
• DNA profiling – capillary electrophoresis

  • Used to identify number of repeating STRs.

  1. DNA template is extracted.
  2. STRs are tagged with different fluorescent markers. Amplified via PCR.
  3. DNA samples ran through capillary electrophoresis.
  4. Computer software is used to determine size of DNA fragment. Larger DNA = more repeating STRs. Smaller DNA = less repeating STRs.
  5. Data is illustrated as electropherogram.
• Bioinformatics

  • The use of ICT and computer software that assists with storage, analysis and communication of DNA sequencing/genome analysis is referred to as bioinformatics.
  • Governments around the world has legislation around this area.
  • Human Genetics Advisory Committee (HGAC) is Australian one.
  • HGAC concerns around bioinformatics is:
  • Whose data is stored on the human genetic database?
  • Who has access to this data?
  • How is this data used?