Nucleic Acid Manipulation Quiz

Nucleic Acid Manipulation - Basic Tools and Enzymes

• Enzymes enable the analysis and manipulation of DNA
• Type II restriction endonucleases are crucial in molecular biology
• These enzymes recognize specific DNA sequences (4-6 base pairs) and cut them in a defined manner
• Palindromic or inverted repeat sequences are recognized
• Cleavage can leave flush-ended or staggered (cohesive-ended) fragments
• Staggered ends are complementary and can anneal to each other
• DNA ligase joins the ends
• Recombinant DNA is widely used in molecular biology
• Enzymes used in molecular biology include specific examples, recognition sequences and products

Isolation and Separation of Nucleic Acids

• DNA isolation involves cell rupture (gentle method to avoid fragmentation)
• EDTA chelates Mg2+ ions, preventing DNase activity
• Cell walls are digested (e.g., lysozyme)
• Cell membrane is solubilized with detergent
• Ribonuclease (RNase) removes RNA
• Proteins are removed with phenol/chloroform
• DNA precipitates with ethanol
• DNA is then redissolved in a buffer
• DNA can be purified using density gradient ultracentrifugation

Isolation and Separation of RNA

• RNA isolation is similar to DNA isolation, but RNase inhibitors are important
• Using guanadinium thiocyanate inhibits RNase and denatures proteins
• RNA is less easily damaged by shearing
• RNA is susceptible to degradation by RNases
• RNA can be isolated from total RNA by using oligo(dT)-cellulose columns

Automated and Kit-Based Extraction of Nucleic Acids

• Automated and kit-based extraction methods for nucleic acids now exist
• These methods are standardized and quality tested
• Automated machines process samples rapidly in microtitre plates
• Nucleic acid extraction is now computer-controlled

Electrophoresis of Nucleic Acids

• Electrophoresis on agarose or polyacrylamide gels separates DNA based on size
• Ethidium bromide stains DNA for visualization under UV light
• Pulsed-field gel electrophoresis (PFGE) separates larger DNA fragments

Automated Analysis of Nucleic Acid Fragments

• Automated systems with pre-cast gels and standardized reagents exist for nucleic acid separation and analysis
• These are helpful where many samples or high-throughput analysis is required
• Automated systems using microfluidic circuits avoid gel preparation

Molecular Analysis of Nucleic Acid Sequences

• Restriction mapping analyzes restriction fragments' size to map DNA
• Electrophoresis of DNA is based on size separation
• Blotting methods (Southern blotting) transfer DNA from gels onto membranes
• DNA probes (labelled) bind to complementary DNA sequences on the membrane
• RNA can be analysed using Northern blotting
• Several methods exist for labelling DNA probes (radioactive or non-radioactive)

PCR (Polymerase Chain Reaction)

• PCR is a method that amplifies a DNA region from a complex source
• Two primers bind to complementary sequences flanking the DNA region
• Cycles of denaturation, annealing and extension amplify the target DNA exponentially
• A thermostable DNA polymerase is used
• PCR has several applications including amplification, detection, analysis of gene expression etc
• Quantitation PCR (qPCR) measures initial DNA/cDNA template concentration

Nucleotide Sequencing of DNA

• Sanger sequencing produces DNA fragments of differing lengths, ending with a ddNTP
• Determining the order of DNA bases
• Several sequencing methods exist including Sanger and Maxam-Gilbert methods
• Maxam-Gilbert method uses chemical reactions to cleave DNA at specific bases
• Sanger sequencing uses dideoxynucleotides to terminate DNA synthesis at each specific base