Nucleic Acid Analysis

Course and instructor: 4BICH001W Biochemistry, Dr Sarah K Coleman
Learning Outcomes:
- Describe the steps and principles of isolating and purifying nucleic acids.
- Explain the underlying principles of basic nucleic acid analysis techniques.
- Provide examples of applications in bioscience research, medicine, and forensics.
Nucleic Acids:
- Methods for extracting and analyzing nucleic acids.
- Manipulation techniques for nucleic acids.
- Types and interpretation of data obtained from nucleic acid analysis.
- Rationale for nucleic acid analysis (DNA to RNA to Protein)
Sample Sources:
- Various biological sources like bone marrow, mitochondria, blood, CSF, saliva, nasal fluid, feces, sewage, soil, water, air, and even a coffee cup.
Applications of Nucleic Acid Analysis:
- Research: gene organization, gene expression, evolutionary relationships, regulation of gene expression, pathologies determination.
- Medicine: diagnosis, treatment, screening, paternity testing, identification, modifying organisms, prevention, diagnosis, treatment, epidemiology.
- Forensic science: familial relationships, identifying human remains, monitoring and tracking endangered species (DNA barcoding), preventing illegal trade.
- Archaeology, Agriculture, Biotechnology.
Extracting and Purifying Nucleic Acids:
- Modern techniques use DNA/RNA binding to silica matrices. Automation via kits is common.
- AIM: release nucleic acid, remove contaminants, enable binding to silica, remove more contaminants, change conditions, collect in a suitable solution.
- What destroys nucleic acids?
Preparation of Samples:
- Grinding, homogenization and enzymes (e.g., lysozyme) to open cells (Plant, Fungi, Bacteria).
- Disrupting membranes (lysing) for mammalian cells.
Extracting Nucleic Acids:
- Protocols involving cell lysis, DNA/RNA binding to silica, changing buffers and removing contaminants. Knowledge of pH, ionic charges, salts, molecular charges and interactions is crucial.
Isolation of Messenger RNA (mRNA):
- Importance in gene expression studies.
- mRNA is less stable than DNA.
- mRNA is converted to complementary DNA (cDNA). cDNA is more stable and readily used in molecular biology.
Reverse Transcription:
- Making DNA from RNA;
- Eukaryotic mRNA has a polyA tail (AAAAAAAAAAAAA). Oligonucleotide primer and Reverse Transcriptase enzyme is needed to convert isolated mRNA to cDNA.
Initial Analysis of Nucleic Acids (Spectrophotometry):
- Techniques for determining the concentration and purity of DNA/RNA.
- DNA and RNA maximum absorb light at λ = 260 nm (UV range).
- DNA concentration: Abs260nm x 50 x dilution factor = µg/mL DNA
- Absorptivity constant is 50 for DNA and 40 for RNA (µg/mL)
- DNA purity is determined by the absorbance ratio of A260/A280. Ratios: 1.8 - 2 = pure, <1.8 = protein contamination, >2.1 = organic contamination.
DNA Manipulation: PCR and amplification:
- Polymerase Chain Reaction (PCR) exponentially amplifies DNA.
- Revolutionized molecular biology, developed in 1980s and Kary Mullis won the Nobel Prize for Chemistry in 1993. Protocols using primers, Taq DNA polymerase, appropriate buffers, and a PCR machine
Site-Directed Mutagenesis by PCR:
- Design oligonucleotides with specific changes in bases.
- Mutagenesis through PCR to increase the amount of mutated DNA.
- Change in amino acid sequence of specific code. (e.g., Tyrosine (codon TAC) to Alanine (codon GCC))
- Original techniques developed by Michael Smith.
Analysing DNA by Electrophoresis:
- Methods for checking DNA presence, size, sequence, and mutations.
- Agarose gel and capillary electrophoresis used.
Capillary Electrophoresis: DNA Sequencing:
- Standard for DNA sequencing.
- DNA molecules move in an electrical field, mobility due to size, detected by UV or fluorescent tags, and data is interpreted by computer.
- Sanger sequencing techniques.
Agarose Gel Electrophoresis: Separation by size through molecular mesh, DNA moves within an electrical field, different agarose percentages in buffer (weight per volume) resolve DNA bands.
Restriction Enzyme (RE) Mapping:
- Restriction enzymes cut DNA at specific places.
- Differently sized fragments are separated.
- Size determination through comparison to a DNA ladder (fragments of known size).
Hybridization Techniques:
- Used for gene expression analysis, identifying variations (mutations).
- DNA microarrays (Current: DNA and RNA analysis). Southern Blots (Old: DNA analysis). Northern Blots (Old: RNA analysis).
DNA Microarray Chips:
- Short oligonucleotides are printed onto a chip.
- Used in a range of studies including gene expression studies or detecting genomic variations.
Comparing Different Tissues:
- Using DNA Microarrays to measure the expression level of genes in different tissue samples via hybridization and detection.
Applications in Bioscience Research: Examining functional gene organization, gene expression regulation, identification of key regulatory genes, genetic influences on diseases (cancer, aging, metabolism), evolutionary relations, identification of human/hominid prehistory and relationships, species identification.
Applications in Medicine: Preventative, diagnostic, and therapeutic applications, genetic testing, tracking prevalence of diseases (pathogens).
Forensics Applications: DNA profiling (fingerprinting), familial relationships analysis, identification of human remains, monitoring and tracking endangered species.
Ethical and safety issues: Important considerations before deploying recombinant DNA technologies.