Dna extraction

• DNA


  A polymer made of nucleotides, which are made of a sugar group, a phosphate group, and a base

  
• Sugar Phosphate Backbone


  The collective name for the components of a nucleotide

  
• Hydrogen bonds


  The bonds that hold DNA strands together, formed between the bases of adjacent strands

  
• Adenine


  Pairs with Thymine
• Guanine


  Pairs with Cytosine
• Coding regions


  Also known as genes, the regions of DNA that contain instructions for making proteins

  
• Exons


  The parts of genes that contain the instructions for making proteins
• Introns


  The parts of genes that do not contain instructions for making proteins

  
• Types of Short Tandem Repeats (STRs)


  • Simple Repeats
  • Compound Repeats
  • Complex Repeats
• Simple Repeats


  • Units of identical length and sequence e.g. TCTA; TCTA; TCTA; TCTA; TCTA; TCTA

  
• Compound Repeats


  • 2 or more simple repeats e.g. TCTA;TAGG; TCTA;TAGG; TCTA;TAGG

  
• Complex Repeats


  • Contain several blocks of variable unit length, along with more or less variable intermediary sequences. E.g. (TTTC)3 TTTT TT*(CT)0-1 CTCC
• Properties of STRs that make them useful for individual human identification


  • Small size (low molecular weight)
  • Polymorphism (Variation in the number of repeating units)
  • Abundant (present throughout the genome)
  • Discrete (Definitive allele identification)
  • Ease of amplification

  
• Polymorphism


  Variation in the number of repeating units in STR sequences

  
• Allele designation


  The number of repeat units displayed by an individual at a particular STR locus
• STRs are distributed abundantly throughout the human genome, occurring randomly approximately every 6 - 10 KB
• STR alleles have discrete designations i.e. whole number of repeat units
• Small size of STR alleles


  • Usually 100-470 bp, which helps maintain integrity during PCR and analysis of small/degraded samples
• Ease of amplification of STRs

  • STRs are amenable to amplification by PCR, which is easy to standardise and automate
• STRs can be co-amplified so that only one test is required
• Factors in selecting STR loci for forensic use


  • Discriminating Power
  • Size range
  • Ability to co-amplify
  • Amplification fidelity
  • Independence
• Discriminating Power


  How common or rare a particular allele is at a given locus, i.e. the potential power of a system to distinguish between individuals
• Size range


  The predicted length of alleles should be between approximately 90 - 500 bp, such that the size range of a candidate locus is less than 340 bp
• Smaller allele sizes afford a higher chance of success with old or degraded samples, and tend to be sized more accurately and reliably
• Ability to co-amplify


  The ability of candidate loci to amplify with other loci efficiently and with minimal problems is essential for multiplex systems
• Amplification fidelity


  Candidate loci should amplify efficiently and exhibit minimal stutter production, clean allele peaks and minimal or no levels of non-specific amplification artefacts
• Independence


  Candidate loci should ideally be located on separate chromosomes to eliminate the possibility of genetic linkage, which can complicate statistical interpretation
• Extraction


  The term extraction covers both the Lysis and Purification stages at the start of DNA analysis
• Factors in choosing extraction method


  • Sample type
  • Quantity of sample type and caseload
  • Method success rate
  • Chemicals required
  • Experience and skills of the analyst performing the work
• Lysis


  The process of breaking down the cell membrane to release the DNA
• Lysis reagents


  A buffer/detergent to lyse the cell membrane, and an enzyme (Proteinase K) to break down proteins
• Proteinase K


  A recombinant protein derived from Pichia Pastoris which is highly specific to cellular breakdown, remains stable over a wide range of temperatures and pH, and breaks down the nuclear membrane by cleaving peptide bonds
• Lysis Theory


  1. Buffer/detergent attacks the cell membrane

  2. Proteinase K incubated at approx. 56oC

  3. Lysate
• Split Preferential Lysis


  A method to obtain and separate DNA from mixtures of sperm and epithelial cells, by lysing the epithelial cells while leaving the sperm cells intact
• Split Preferential Lysis


  1. Epithelial cells are lysed by SDS buffer

  2. Sperm cells pellet due to centripetal force

  3. Epithelial lysate is removed

  4. Sperm cells are washed

  5. DTT is added to lyse sperm cells
• DTT


  Dithiothreitol, a reducing agent that breaks down the disulphide cross-links in the protamine surrounding the sperm head
• Bone and teeth


  Contain a large number of disulphide bridges in the nucleases, making them resilient to standard lysis buffers
• Bone and teeth extraction


  1. Need to be ground to a powder form

  2. Require longer incubation periods at lysis

  3. Minerals in bone can act as inhibitors so need to be removed during purification
• Purification


  The process of removing unwanted cell material after lysis, to obtain purified DNA
• Purification methods


  • Silica coated beads
  • Spin column