Nucleic Acid Synthesis and Types

Nucleic Acid Synthesis Direction

• Nucleic acid synthesis proceeds in the 5' to 3' direction.

DNA Types

• Genomic DNA
• Mitochondrial DNA
• Chloroplast DNA
• Plasmids
• PCR products
• cDNAs
• Primers
• Oligonucleotides

Plasmids

• Extrachromosomal, circular DNA
• Self-replicates
• Found in yeast and bacteria
• Used in research

DNA Isolation Steps

• Cell lysis: Dissolve cell membrane using detergent
• Protein denaturation/removal: Use protease or cation-chelating beads
• DNA concentration/precipitation: Centrifuge or alcohol precipitation

DNA/RNA Concentration Measurement

• UV absorbance spectroscopy (ng/µL)
• Real-time PCR

Oligonucleotides (Oligos)

• Synthetic DNA
• Used for plasmid construction, RNA interference (RNAi), PCR primer design, and hybridization probes

Polymerases

• Enzymes that add nucleotides in the 5' to 3' direction

Polymerase Types

• Taq polymerase: DNA-dependent DNA synthesis
• Reverse transcriptase: RNA-dependent DNA synthesis
• RNA polymerases: DNA-dependent RNA synthesis

Primers

• Short (20-30 nucleotides) DNA sequences
• Define the target sequence for DNA polymerase
• Anneal at temperature dependent on sequence

Restriction Endonucleases

• Cut DNA at specific sequences
• Can create sticky ends (5' overhangs) or blunt ends

Gel Electrophoresis

• Separates DNA fragments based on size (smaller fragments move faster)
• DNA migrates from the cathode (-) to the anode (+) due to its negative charge
• Agarose or polyacrylamide gels

Gel Electrophoresis Stains

• SYBR Safe: DNA visualization under UV light
• Fast Blast: DNA visualized as blue

Hybridization

• Base pairing between complementary nucleic acid sequences

Tm (Melting Temperature)

• Temperature at which 50% of primers bind to genomic DNA
• Equilibrium between melting and annealing

Tm Formula

• Tm = 4(C+G) + 2(A+T)
• Note:* C and G are cytosine and guanine; A and T are adenine and thymine; and bp is base pair.

Probe

• Short, labeled nucleic acid sequence
• Used to detect a specific DNA sequence in a sample

Hybridization Steps

• Immobilize nucleic acids
• Melt DNA strands
• Hybridize under increasing stringency
• Detect probe (radioactively labeled or fluorescently labeled)

Southern Blot

• Detects DNA sequences on a membrane using a DNA probe

Northern Blot

• Detects RNA sequences on a membrane using a DNA probe

Western Blot

• Detects proteins on a membrane using antibodies

PCR (Polymerase Chain Reaction)

• In vitro DNA replication
• Exponential increase in DNA fragments
• Uses genomic DNA, primers, dNTPs, buffer, and Taq polymerase

PCR Steps

• Denaturation (94°C): Separate double helix
• Annealing (50-60°C, Tm-dependent): Primer hybridization to genomic DNA
• Extension (70°C): DNA replication

PCR Refinements (No Bands)

• Lower annealing temperature
• Increase MgCl₂ concentration
• Adjust genomic DNA amount

PCR Refinements (Too Many Bands)

• Raise annealing temperature
• Decrease MgCl₂ concentration
• Adjust genomic DNA amount
• Use nested primers

Nested Primers

• Increase PCR specificity

RT-PCR (Reverse Transcription PCR)

• Reverse transcription of mRNA to cDNA using reverse transcriptase and one primer
• PCR using the cDNA and nested primers

mRNA Feature

• Poly-A tails
• Amplified using oligo dT primers

qPCR (Quantitative PCR)

• Measures amount of starting DNA in real time
• Uses modified thermocycler

Ligase

• Repairs phosphodiester bonds using ATP
• Joins blunt or sticky ends

Molecular Cloning

• Isolating and replicating a DNA fragment in another organism

Cloning Vector Parts

• Origin of replication
• Antibiotic resistance gene
• Unique restriction sites
• Screenable marker

Screenable Marker

• Enzyme that converts a colorless substrate to a colored product or a second antibiotic gene
• Allows for selection of cells containing the insert

Transformation

• Introducing foreign DNA into a cell

Transformation Steps

• Prepare cells (make them competent)
• Incubate cells with plasmids
• Shock cells
• Allow cells to recover

Transformation Methods (Artificial)

• CaCl₂ and heat shock
• Electroporation
• Microinjection

Transformation Methods (Natural)

• DNA uptake from environment
• Conjugation
• Transduction

Restriction Digest

• Cutting DNA at specific locations using endonucleases

Sanger Sequencing

• Uses Taq polymerase, primers, dNTPs, fluorescently labeled ddNTPs
• Separates products by electrophoresis
• Detects DNA sequence using a laser

Site-Directed Mutagenesis

• Precise DNA modifications using mutagenic primers and PCR

Expression Vectors

• Contain transcription and translation initiation/termination sequences

Yeast Transformation Advantages

• Eukaryotic
• Easy to grow
• Natural plasmids
• Well-characterized genome

Plant Transformation Challenges

• No origin of replication
• Cell walls

Plant Transformation Methods

• Biological (Agrobacterium)
• Physical (biolistics)

Plant Model Organism

• Arabidopsis thaliana

Plant Transformation Advantages

• Totipotent cells (can differentiate)

GM Crop Advantages

• Breeding out undesired traits (allergens, unhealthy components)
• Insect resistance (reduced pesticide use)
• Nutritional enhancements (e.g., Golden Rice)

GM Crop Concerns

• Possible antibiotic resistance
• Gene transfer to other plants
• Environmental damage

BLAST

• Basic Local Alignment Search Tool

Animal Cell Transformation Advantages

• Specific protein folding/modifications

Commonly Transformed Animal Cell Lines

• Transient: COS, BHK, HEK-239
• Stable: CHO

CRISPR-Cas9

• Bacterial defense mechanism adapted for genome editing

Ex Vivo Gene Therapy

• Correcting gene sequences in removed cells and returning them to the body

In Vivo Gene Therapy

• Delivering corrected gene sequence directly into the body

Genomic Libraries

• Collection of all an organism's DNA fragments

cDNA Libraries

• Collection of all an organism's genes

Bacterial Artificial Chromosomes (BACs)

• Vectors for cloning large DNA fragments (100-300 kb)

Next Generation Sequencing (NGS)

• Massively parallel sequencing
• Uses physical separation and PCR
• Advanced light detection
• High-performance computing

Gene Expression Measurement Methods

• Destructive: Northern Blot, RT-qPCR, Microarray, RNAseq
• Non-destructive: In situ hybridization, Promoter assays

Microarrays

• cDNAs or gene fragments attached to a support
• Fluorescently labeled samples hybridize to the support

In Situ Hybridization

• Detects gene expression within tissues using a DNA probe

Antibody-Based Protein Detection Methods

• Western Blotting
• ELISA
• Immunoprecipitation/co-immunoprecipitation
• Immunohistochemistry

This quiz covers key concepts in nucleic acid synthesis, including the direction of synthesis, types of DNA, and important processes like DNA isolation. Additionally, it explores the role and types of polymerases involved in these biochemical reactions. Test your understanding of these foundational topics in molecular biology.