Molecular Methods in Drug Discovery

• This lecture covers the basic principles, applications, and health and safety considerations of molecular methods in drug discovery.
• Techniques discussed: DNA/RNA extraction and purification, DNA sequencing, basic DNA cloning, PCR & RT-PCR.

DNA Structure

• DNA has a double helix structure with major and minor grooves.
• DNA consists of:
  • Deoxyribose: A Pentose sugar
  • Adenine (A) and Guanine (G): Purines
  • Thymine (T), Cytosine (C), and Uracil (U): Pyrimidines

DNA Components

• Nucleosides or deoxynucleosides form from purine and pyrimidine bases and either ribose or deoxyribose.
• Nucleotides or deoxynucleotides form from nucleosides/deoxynucleosides and phosphoric acid.
• Nucleic acid (DNA and RNA) is then produced.
• Adenine (A) pairs with Thymine (T), and Guanine (G) pairs with Cytosine (C).

Sources of DNA

• Blood, semen, saliva, and urine
• Hair (with root and shaft), teeth, bone, and tissue
• Plasma, cigarette butts, envelopes & stamps, and fingernail clippings
• Chewing gum, bite marks, and faeces

DNA Recovery

• A single diploid cell contains about 6 picograms of DNA.
• There is approximately 5-10 x 10^6 white blood cells/ml of blood.
• Expect approximately 30-60 ng of DNA per microliter of blood.
• A PCR reaction requires about 1 ng of DNA or 10^2-10^5 cells.

DNA Extraction

• The most common DNA extraction procedures in molecular biology include:
  • Organic (phenol-chloroform) extraction
  • Non-organic (proteinase K and salting out)
  • Solid phase extraction (spin column)
  • FTA paper
• The method used depends on sample, technique, or analyst preference.
• Organic extraction involves lysing the cell membrane, keeping nuclei intact, and pelleting nuclei.
• Sodium dodecyl sulfate + proteinase K is then added to resuspend nuclei, lyse the nuclear membrane, and digest protein.
• Phenol-chloroform is used to extract released DNA and remove proteinaceous material.
• Protein contaminants are denatured and partition either with the organic or at the interface between organic and aqueous phases.
• Nucleic acids remain in the aqueous phase.
• Add ice-cold 95% ethanol and a salt (e.g., sodium acetate) to precipitate DNA from the aqueous layer.
• Wash precipitated DNA with 70% ethanol, dry under vacuum, and resuspend in Tris-EDTA buffer.
• Non-organic DNA extraction doesn't use organic reagents like phenol or chloroform.
• Digested proteins removed via salting out with high concentrations of LiCl.
• Salts that aren't adequately removed can cause problems in downstream procedures due to altered DNA mobility (band shifting).
• Solid Phase Extraction is kit-based.
• DNA is immobilised on a solid matrix, proteins are washed out, and then DNA is eluted.
• FTA paper contains a unique mixture of strong buffers, protein denaturants, chelating agents, & a UV-absorbing, free radical trap.
• Reagents are impregnated into a cellulose-based filter matrix, such as Whatman filter paper.

Functions of FTA paper

• Kills blood-borne pathogens and lyses cells on contact.
• Immobilizes DNA within the matrix, protecting it from degradation.
• Allows for long-term storage at room temperature.
• FTA paper is used to conduct neonatal heel blood spot testing usually on day 5 for:
  • Sickle cell
  • Cystic fibrosis
  • Congenital hypothyroidism
  • 6 inborn errors of metabolism
  • phenylketonuria (PKU)
  • medium-chain acyl-CoA dehydrogenase deficiency (MCADD)
  • maple syrup urine disease (MSUD)
  • isovaleric acidaemia (IVA)
  • glutaric aciduria type 1 (GA1)
  • homocystinuria (HCU)
• At home, DNA extraction uses strawberries and peas and is a non-lab based approach.

Types of DNA Manipulation

• Visualize DNA using agarose gel electrophoresis.
• Cut up or join DNA back together through restriction enzyme digestion.
• Make more of the DNA by polymerase chain reaction (PCR) or cloning.
• Read the DNA via sequencing.

Separating DNA by Size

• Used by Agarose gel electrophoresis separates DNA by size.
• Use 1-4% agarose is used for separating fragments 0.2-20 kbp (kilobase pairs)
• Due to phosphate groups, DNA is negatively charged, migrates towards anode (+).
• Run the dye to red
• DNA is loaded into wells cut in the gel.
• A fluorescent dye is added to the gel: EtBr or SYBr.
• Fluoresces under UV irradiation due to interaction with the DNA.
• A loading dye (usually blue) is added to the sample to keep the DNA in the well and track its migration.
• Molecular weight standards are also run.
• Gel extraction involves cutting out the gel slice with your band, releasing the DNA from the gel, adding it to a spin column, and extracting DNA as before.

Molecular Methods

• Used to visualise the DNA via Agarose gel electrophoresis
• Used together or cut it up or join it back via Restriction enzyme digestion
• Also used to make more of it via Polymerase chain reaction (PCR) and Cloning
• And finally to Read the DNA via Sequencing

Enzymes

• Enzymes that interact with nucleic acids:
  • Nucleases and restriction enzymes for Digest (cut) DNA or RNA
  • Ligases for Join breaks in DNA
  • Polymerases for Synthesises DNA or RNA. Also used in Reverse transcriptase (RNA to DNA)
• Deoxyribonuclease (DNase) digests DNA (double strandase).
• Ribonuclease (RNase) digests RNA (single strandase).
  • Endonucleases breaks internal phosphodiester bond
  • Exonucleases sequentially remove nucleotides from the end of the molecule
• Restriction endonucleases cut DNA at specific sequences and are used in molecular biology.

Restriction Enzymes

• Enzymes for Serratia marcescens: Smal
• Enzymes for Escherichia coli: EcoRI
• Each bacterium produces a unique enzyme that recognises a specific sequence, the RECOGNITION SITE.
• RECOGNITION SITE may be of 4, 5, 6, 7 or 8 bases.
• This sequence is termed PALINDROMIC because the 3' and 5' seqs are the same on each strand
• Examples of PALINDROMIC:
  • A
  • Wow
  • Racecar
  • God's dog
  • Madam, I'm Adam
  • A Toyotas a Toyota
  • A man, a plan, a canal: Panama
• The enzyme makes a cut within this site at a specific point, the CUTTING SITE.
• Types of ends:
  • Sticky: BamHI and Pstl
  • Blunt: Hpal

Restriction Fragment Length Polymorphism

• RFLP is a difference in DNA sequences that can be detected by the presence of fragments of different lengths after digestion with specific restriction enzymes
• As a molecular marker, RFLP is specific to a single sequence/restriction enzyme combination.
• Disease diagnosis and susceptibility can be assessed, as well as Pharmacogenetics