Lecture 1 - NA Isolation & PCR Flashcards
What is rDNA?
Recombinant DNA - artificially created, genetic sequences not usually found together in nature
What are the 6 steps in molecular cloning?
- Isolation of target gene
- Insertion into vector DNA
- Transfer into microbes via transformation/infection
- Selection of microbial cells containing desired recombinant vectors
- Growth of transformed microorganisms
- Expression of the gene to obtain product
Describe the Central Dogma
DS DNA transcription mRNA copy of one strand translation Amino acid sequence of protein
What are the aims when isolating target nucleic acid?
High quantity, quality and integrity
What are the 3 steps of isolating the target nucleic acid?
- Disruption of cell membrane/cell wall
- Separation and precipitation of nucleic acid
- Dissolving nucleic acid
What is cell lysis?
Outer boundary of cell membrane is broken down to release inter-cellular materials (e.g. DNA, RNA, protein or organelles)
What are the 2 types of cell lysis methods?
Mechanical and non-mechanical
What influences the lysis method chosen?
- Ease of purification steps
- Target molecules for analysis
- Quality of final product
In mechanical lysis, cell membrane is physically broken down using shear force. What are the 2 types of mechanical lysis?
- High pressure homogeniser - cells in media forced through orifice valve using high pressure, disruption of membrane occurs due to compression and expansion
- Bead milling - cells disrupted by tiny beads at high speeds
Very efficient method to lyse a wide range of cells
Limitation: Heating, degradation of cellular products and higher cost
Non-mechanical lysis
What are the physical methods?
- Freeze thaw cell lysis - ice helps break down cell membrane (can be time consuming, cannot be used for temperature sensitive cellular components)
- Ultrasonic cavitation - shockwave disintegrates cell membranes, disruption is independent of biomass concentration (but large amount of heat generated)
- Osmotic shock
Non-mechanical lysis
What are the chemical methods?
- Alkaline lysis - OH- ion in NaOH breaks fatty acid-glycerol ester bonds in cell membrane, sodium dodecyl sulphate (SDS) solubilises the proteins and membrane (mostly used for isolating plasmid DNA from bacteria)
- Detergent lysis - disrupt hydrophobic-hydrophilic interactions of cell membranes, breaking the structure of water and making it less hydrophilic (chaotropic agents e.g. urea, guanidine and EDTA can also be used)
Non-mechanical lysis
What is biological cell lysis?
Enzymes such as lysozyme, lysostaphin, zymolase, cellulase, protease, or glycanase are used
Highly specific
Lysozyme are used for bacterial cell lysis by specifically hydrolysing glycosidic bonds in peptidoglycan layers
Chitinases for yeast cell lysis and pectinases are used for plant cell lysis
What are the methods of isolating nucleic acid?
- TRIzol reagent (covered in practical class)
- Silica-based extraction
- Magnetic beads
- Caesium chloride method
Methods of isolating nucleic acid
What is silica-based extraction?
Negatively charged nucleic acids interact with positively charged silica membrane and are retained
Rest of the unbound soluble material filters through membrane
Impurities and loosely bound molecules are removes using washing buffer
DNA bound is eluted by dissolving in buffer and collecting
Methods of isolating nucleic acid
How are magnetic beads used?
Positively charged magnetic beads entrap negatively charged DNA/RNA
DNA/RNA loaded beads are concentrated and separated from solution using a magnetic field
DNA/RNA can then be precipitated using ethanol
Methods of isolating nucleic acid
How is caesium chloride used?
Molecular weight is used to extract DNA/RNA
Density gradient of CsCl is established in a centrifuge tube
Nucleic acid settles down as a band in the gradient where density of DNA and gradient is the same
What to do if gene sequence of gene of interest is not available?
- Construction of genome/cDNA library
2. Screening of gene of interest such as protein product, enzyme function
What to do if gene sequence of gene of interest is available?
PCR amplification of target gene
How is genome library constructed?
Genomic DNA isolated digested by restriction enzymes
Vector plasmid digested with restriction enzyme
Digested genomic DNA and vector are ligated together and transformed into bacterial host cells
Recombinant plasmid generated is subsequently transformed into a host cell for amplification
How to generate cDNA from eukaryotic genes?
Reverse transcription of mRNA (to obtain functional sequence of gene)
Use oligo(dT) primer for polyA tail, and reverse transcriptase enzyme
RNA strand then hydrolysed by reverse transcriptase enzyme
What are the 3 steps in PCR?
- Denaturation - high temperature (90-100°C), separation of double DNA helix into two strands
- Annealing - quick cooling (50-65°C), two primers bind to their complementary sequences on the template DNA strands
- Extension - the reaction mixture is heated again (60-70°C), DNA polymerase extend the primers by adding complementary dNTPs
How is PCR used in infectious disease diagnostics?
RNA extracted from swab samples using TRIzol reagent
RNA transcribed into cDNA and specific regions of interest amplified by PCR
Primers used target the sequences coding for RNA dependent RNA polymerase (RdRP), Spike protein (S), Envelope protein (E) and Nucleocapsid protein (N)
How is PCR used in environmental science?
Environmental DNA (eDNA) metabarcoding is a method of assessing biodiversity
DNA from samples are taken from the environment via water, sediment or air
Primers used generally target the genes such as Cytochrome oxidase-I (COI), Ribulose biphosphate carboxylase large chain (rcbL) and 12s or 16s ribosomal RNA
How is PCR used in forensic science?
The number of repeated nucleotides in hypervariable microsatellite sequence (VNTR - variable number of tandem repeat) is used to distinguish whether sample at the crime scene matches that of the suspect
Primers designed to prime at specific VNTR loci in homologous chromosomes
