Biotechnology pt2

• Cloning


  Making a genetically exact copy of something
• DNA cloning


  • Cloning a gene or other small piece of DNA

  
• Recombinant DNA


  1. Gene for Human insulin inserted into Bacterial cell

  2. DNA recombination
• DNA library


  A collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning

  
• cDNA library


  A collection of only the genes that are encoded into proteins by an organism (exon)

  
• Genomic DNA library


  A collection of DNA fragments that make up the full-length genome of an organism (introns and exons)

  
• Creating a genomic DNA library

  1. DNA is digested with restriction enzyme

  2. DNA fragment is ligated in a plasmid

  3. Bacteria is transform (insertion of the plasmid into the bacteria)

  4. Bacteria multiply (cloning the plasmid)

  
• Genomic libraries


  • Less useful for eukaryotic cells, since DNA contains introns, thus the gene cannot be expressed by bacteria
  • Played an important role in the whole genome sequencing of several organisms, including the human genome and several model organisms
  • Now mostly obsolete due to the advance in modern sequencing technique that do not need DNA genomic library

  
• Finding a gene in a DNA genomic library

  1. Southern blot

  2. PCR

  
• Creating a cDNA library

  1. m-RNA is reverse transcribed into c-DNA

  2. c-DNA fragment is ligated in a plasmid

  3. Bacteria is transform (insertion of the plasmid into the bacteria)

  4. Bacteria multiply (cloning the plasmid)

  5. c-DNA is them amplified using PCR

  
• cDNA library


  • Represent a stable version of the m-RNA expression at the moment of sampling
  • Useful since they don't contain intron, so Eukaryotic gene can be express in Bacteria

  
• Producing insulin using biotechnology

  1. Do a RT-PCR on the m-RNA of insulin

  2. DNA amplification (PCR, purification)

  3. DNA ligation into a vector (e.g. plasmid)

  4. Transformation of the host cell with recombinant DNA

  5. Cloning of host cells

  6. Detection of selected cells that contain the insulin gene
• DNA fingerprinting


  The use of biotechnology in forensic science to identify criminals or do paternity tests
• RFLP DNA fingerprinting


  1. Fragments GENOME by cutting with restriction enzymes

  2. DNA fragments are migrated on a electrophoresis gel to separate the DNA by size

  3. The band pattern is compared

  
• STR DNA fingerprinting


  1. PCR is used to amplify the STR locus (primers flanking the locus)

  2. The amplified DNA is migrated on an electrophoresis gel
• STR DNA fingerprinting


  • Short tandem repeats (STRs), typically 2 to 4 nucleotides long, are not part of coding or regulatory regions of genes and mutate over generations so that the length of the repeats varies
  • Each person has a different number of repeats at various locations (loci) in their genome, therefore the size of each person's STRs is different
  • More than one locus is analysed to add to the accuracy to the method

  
• DNA sequencing is the process of determining the order of nucleotides in a DNA molecule

  
• Short tandem repeats (STRs)

  Regions of DNA where a short sequence of nucleotides is repeated consecutively, the number of repeats of each person's STRs is different, which can be used as a tool for identification
• DNA fingerprinting using STRs

  1. PCR is used to amplify the STR locus (primers flanking the locus)

  2. The amplified DNA is migrated on an electrophoresis gel

  3. STR method have the advantage of needing only a SMALL sample of DNA, since the DNA is amplified by PCR
• DNA fingerprinting using STRs

  • More than one locus is analysed to add to the accuracy to the method
  • STRs have generally 2 bands, since humans have pairs of chromosomes and thus pairs of STRs
  • One STR comes from the mother and the other from the father
  • If only one band is visible, this means that the mother and father had the same number of repeats on their STRs
• DNA sequencing


  The process of determining the order of nucleotides (the four bases: adenine, guanine, cytosine, and thymine) in DNA

  
• Types of DNA sequencing

  • Sanger method
  • Automated methods
• Sanger method


  Labour intensive, based on DNA replication (PCR) where copied fragments end with a chain terminating nucleotide called dNTP, produces nested fragments whose sequence is determined by gel electrophoresis

  
• Automated DNA sequencing methods


  Way faster than the traditional Sanger method
• Sanger method history


  The first automated method was based on a technique called Dideoxyribonucleotide chain termination method, developed by Frederick Sanger who received a Nobel Prize in 1980 for this achievement
• Sanger method


  1. Similar to PCR method, which needs primers, free nucleotides (dNTP), Taq polymerase III

  2. DNA Polymerase III requires a 3'-OH to add nucleotides during DNA replication

  3. Some nucleotides (ddNTP) do not have 3' –OH and thus no other nucleotide can be added after which terminates the sequence

  4. There is a mix of normal dNTP that can continue the amplification and ddNTP that terminate it, creating an array of different size fragments that got stop randomly from amplifying at different lengths

  5. In the manual Sanger method the ddNTP were radioactive, and the amplified DNA was migrated on a gel and exposed to a film to reveal the radioactive ddNTP

  6. In the newer automatic Sanger method, 4 different colours of fluorescent ddNTP are used and the gel electrophoresis is done in a capillary, where each fluorescent ddNTP is scanned by a laser and the sequence is automatically generated

  
• Next generation sequencing


  Do not have an electrophoresis gel, DNA is cut and attached to a solid surface, DNA is copied by PCR to turn one molecule of DNA into a cluster of identical DNA pieces, then a PCR is made with special fluorescent nucleotides which prevent other nucleotides from being added, and a picture of the flow cell is taken to determine the sequence

  
• The Human Genome Project (HGP) was an international scientific research project lasting 11 years from 1990 to 2001, designed to determine the sequence of nucleotide base pairs and identify and map all of the genes in the human genome

  
• The total cost of the first human genome was between $500 million to $1 billion, but with the advent of new technology the cost is consistently dropping, and now the complete genome can be done for as low as $1,500 US (in 2015)
• Sequencing gives you thousands of sequences of fragments of the genome, and those fragments sequences are overlapping, so the whole sequence can be recreated by assembling them like a big puzzle

  
• Genomes that have been sequenced

  • Chimpanzee
  • E. coli and many other prokaryotes
  • Zea mays (corn)
  • Drosophila melattogaster (vinegar fly)
  • Mus musculus (common mouse)
  • Macaca mulatta (rhesus macaque)
  • Homo neanderthalensis (Neanderthal)
• The proportion of genetic material inherited from Neanderthals is approximately 1.5 to 2.1%
• CRISPR-Cas9


  A gene editing tool that consists of two parts: the Cas9 enzyme that unwinds and cuts DNA, and the guide RNA that recognizes the sequence of DNA to be edited in the genome

  
• How CRISPR-Cas9 works


  1. When foreign viral DNA enters the cell, a Cas complex recognizes it and splits it into small fragments, one of which is added to the CRISPR array in the bacteria genome

  2. The CRISPR array is then transcribed into a long RNA, which is cut into small pieces representing a specific virus

  3. These small RNAs direct the Cas complex (nuclease) to the foreign DNA according to the specificity of the sequence, and if the specific DNA is recognized, it is cleaved, thus eliminating the virus DNA

  
• CRISPR-Cas9 can be used to delete a portion of a gene to inactivate it, insert a portion of DNA in a gene, or correct a portion of a gene using a donor DNA as a template
• CRISPR-Cas9 can also be modified to replace the endonuclease with an activator or repressor to control gene expression, or to express a fluorescent protein to visualize where the sequence of the gene is in the genome