BIOC2371-LECTURE 1

• DNA melting temperature (Tm)

  The temperature at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands

  
• DNA isolation


  1. Disruption

  2. Lysis (may include RNAse)

  3. Removal of proteins and contaminants

  4. Recovery of DNA
• Cellular components


  • Organelles
  • Lipids
  • Proteins
  • Carbohydrates
  • DNA
  • Genomic
  • Single stranded DNA
  • Vector DNA
  • RNA

  
• Cellular processes important to molecular biologists

  1. Replication

  2. Transcription

  3. RNA splicing

  4. Translation

  5. Protein functions

  
• DNA


  Double-stranded molecule consisting of a long chain of nucleotides

  
• RNA


  Usually is a single-strand helix consisting of shorter chains of nucleotides
• DNA


  • deoxyribose sugar
  • phosphate backbone
  • adenine, guanine, cytosine, thymine bases
  • AT (adenine-thymine)
  • GC (guanine-cytosine)
  • Stable under alkaline conditions
  • Minimal space for enzymes to attach

  
• RNA


  • ribose sugar
  • phosphate backbone
  • adenine, guanine, cytosine, uracil bases
  • AU (adenine-uracil)
  • GC (guanine-cytosine)
  • NOT stable under alkaline conditions
  • Large grooves makes more susceptible to enzyme attack
  • Constantly produced, used, degraded, and recycled
  • Relatively resistant to UV damage

  
• DNA denaturation


  1. Heating

  2. Chemical
• Hyperchromic shift


  Abrupt increase in A260 absorbance when DNA melts from double-stranded to single-stranded

  
• Melting temperature (Tm)


  Temperature at which 50% of DNA molecules are denatured

  
• Factors affecting Tm


  • Concentration of DNA
  • Concentration of ions in solution (Mg+, K+, Na+)
  • DNA sequence
  • Length of DNA
• Fluorescence melting curve analysis

  Double stranded DNA specific dye (SyBr Green) will fluoresce when bound to DNA, denaturation to single strands results in loss of fluorescence

  
• Chemical denaturation methods


  • pH
  • Competitive denaturants (e.g. urea, formamide)
  • Covalent modification denaturants (e.g. formaldehyde, glyoxal)
• DNA isolation


  Separation of DNA from cellular components in 4 stages: disruption, lysis, removal of proteins/contaminants, recovery of DNA
• DNA concentration ([DNA])


  Estimated by measuring absorbance at 260nm, multiplying by dilution factor, and using the relationship that A260 of 1.0 = 50µg/ml pure dsDNA
• DNA yield


  Total yield obtained by multiplying the DNA concentration by the final total purified sample volume
• DNA purity


  Estimated by measuring A260/A280 ratio, good quality DNA has a ratio of 1.7-2.0
• Common sources of nucleic acids

  • Whole blood
  • Hair
  • Sperm
  • Bones
  • Nails
  • Tissues
  • Blood stains
  • Saliva
  • Buccal swabs
  • Epithelial cells
  • Urine
  • Stool
  • Paper cards
  • Paraffin embedded
  • Bacteria
  • Animal tissues
  • Wood
  • Plants
• Types of molecules targeted

  • Genomic DNA
  • Organelle DNA (mitochondria)
  • Vector/Plasmid DNA
  • mRNA

  
• Extraction: Disruption and lysis

  Destruction of protein structures to release nucleic acids, using salt solutions, detergents, proteases
• Extraction methods must be adapted to accommodate source and sample size, e.g. fresh vs stored, sample type
• TT with Proteinase K

  1. Centrifuge: discard pellet and retain supernatant

  2. Supernatant (lysate) used to extract DNA
• Feces


  1. Feces are centrifuged. The supernatant is discarded, and the pellet is washed with TE (Tris, EDTA, pH 8) and retained after centrifugation

  2. Buffer containing TNE (Tris-Cl, 0.5% SDS, CaCl2) and Proteinase K is added to pellet and incubated at 55◦C 1-2 hrs (lysate)

  3. Lysate centrifuged to pellet fecal particles (discarded) and potato starch added to supernatant for suspension. Starch reduces the quantity of bile acids and bile salts (e.g. inhibits PCR)

  4. Centrifugation pellets the starch (discarded), the supernatant retained

  5. NaCl and CTAB solution (NaCl, 10% CTAB) added to supernatant and incubated at 70◦C for 10 min (sample)

  6. Extract DNA with phenol:chloroform:isoamyl alcohol
• Paraffin embedded tissues


  1. Subjected to xylene treatment which dissolves the paraffin from the tissue

  2. Rehydrate the sample by using a series of ethanol washes (100% ethanol; 70%; 50%)

  3. Proteins and harmful enzymes such as nucleases are digested by proteinase K in a lysis buffer also containing denaturing agents such as SDS
• Whole blood samples


  1. Blood samples collected and stored at -70oC in EDTA vacutainer tubes

  2. When thawed a standard red cell lysis buffer is added. Mixed gently and centrifuged: supernatant discarded, and pellet retained. This step is done multiple times to remove hemoglobin

  3. Pellet is resuspended in a lysis buffer containing SDS detergent and proteinase K, and incubated at 55oC 1 hr

  4. Sample can be now used for phenol/chloroform/isoamyl alcohol DNA extraction
• Urine


  1. Addition of different preservatives such as guanidine thiocyanate lysis buffer (denature of DNAses and RNAses)

  2. Lysis buffer containing SDS detergent and proteinase K, is added then, and incubated at 55oC 1 hr

  3. Sample can be now used for phenol/chloroform/isoamyl alcohol DNA extraction
• Vector/Plasmid DNA isolation


  1. Liquid culture centrifuged: pellet

  2. Re-suspended cells in buffer containing EDTA (chelating agent)

  3. Disruption of the plasma membrane to release the DNA out of the cell with lysis buffer containing SDS
• Vector/Plasmid DNA isolation


  1. Growth of the bacterial cell

  2. Harvesting and lysis of the bacteria

  3. Purification of the plasmid DNA. Similar to extraction of genomic DNA but separation of genomic from plasmid DNA is necessary (e.g. Ethidium bromide-cesium chloride density gradient centrifugation)
• DNA Extraction methods


  • Salting-out methods
  • Organic extraction methods
  • Anion-exchange methods
  • Silica-based methods
  • Cesium chloride density gradients
• Salting-out methods


  Use of crude lysate, proteins and other contaminants are precipitated from the cell lysate using high concentrations of salt such as potassium acetate or ammonium acetate
• Organic extraction methods


  Uses organic solvents to extract contaminants from cell lysates, cells are lysed using a detergent, mixed with phenol/chloroform and isoamyl alcohol
• Anion-exchange methods (CHELEX)


  Interaction between negatively charged phosphates of the nucleic acid and positively charged surface molecules on substrate, DNA binds to the substrate under low-salt conditions, impurities (RNA, cellular proteins, and metabolites) are washed away using medium-salt buffers, high-quality DNA is eluted using a high-salt buffer and recovered by alcohol precipitation
• Silica-based methods — DNeasy Tissue Kits

  Selective adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of chaotropic salts (denature of DNAses and RNAses), cellular proteins and contaminants washed away, ready-to-use total DNA is eluted from silica-gel membrane using a low-salt buffer
• Cesium chloride density gradients

  Cells are lysed using a detergent, the lysate is alcohol precipitated, resuspended DNA is mixed with CsCl and ethidium bromide and centrifuged for hours, DNA band is collected from centrifuge tube, extracted with isopropanol (removal of ethidium bromide), and DNA is precipitated with EtOH
• RNA (Ribonucleic acid) is a long single-stranded chain of phosphate and ribose units with nitrogen bases adenine, guanine, cytosine and uracil bonded to the ribose sugar, present in living cells and many viruses

  
• RNA Isolation


  1. Homogenization: Tissues - Homogenize in TRIZOL reagent, Cells grown in Monolayer - Rinse with PBS and lyse with TRIZOL, Cells Grown in suspension - Centrifuge, resuspend in PBS, pellet and lyse with TRIZOL

  2. Phase Separation: Phenol, Chloroform and guanidinium thiocyanate added, centrifugation results in 3 phases - top aqueous (RNA), middle interphase (DNA), bottom organic phase

  3. RNA Precipitation: RNA from aqueous phase precipitated with isopropyl alcohol

  4. RNA Wash: Pellet washed with 75% ethanol

  5. RNA re-dissolving: Pellet dissolved in DEPC-treated water/TE buffer
• Concentration and Yield of RNA

  Spectrophotometric analysis: Dilute 1 μl of RNA with 39 μl of DEPC-treated water/TE buffer, take OD at 260 nm and 280 nm to determine sample concentration and purity, A260/A280 ratio should be above 1.6, 1 OD at 260 equals 40 µg /ml RNA
• Protein Purification techniques


  • Based on molecular size: dialysis and ultrafiltration, density gradient centrifugation, size-exclusion chromatography
  • Based on solubility of proteins: isoelectric precipitation, salting out
  • Based on electric charge: ion-exchange chromatography, electrophoresis
• Dialysis and ultrafiltration


  Procedures that separate proteins from small solutes, membrane enclosing the protein solution is semipermeable, allowing the exchange of water and small solutes but not proteins