Nucleic Acid Synthesis and Types

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Questions and Answers

What is the correct direction for nucleic acid synthesis?

5' to 5'

3' to 3'

3' to 5'

5' to 3' (correct)

Which type of DNA is found in both plants and bacteria?

Plasmid DNA (correct)

Chloroplast DNA

Mitochondrial DNA

Genomic DNA

What is the role of primers in DNA replication?

To provide orientation for DNA polymerase (correct)

To visualize DNA in gel electrophoresis

To cut DNA at specific sequences

To denature proteins during isolation

Which enzyme is responsible for adding nucleotides in the 5' to 3' direction and synthesizing DNA?

Taq polymerase Signup and view all the answers

Which of the following substances is used for visualizing DNA in gel electrophoresis?

SYBR Safe Signup and view all the answers

What is the primary function of CRISPR-Cas9 in modern applications?

To manipulate genomic DNA Signup and view all the answers

Which of the following best describes cDNA libraries?

Collections of all genes made from mRNA Signup and view all the answers

Which technique is non-destructive for measuring gene expression?

In situ hybridization Signup and view all the answers

What type of cell line is commonly used for stable transformation?

CHO Signup and view all the answers

Which technique uses molecular separation followed by PCR to produce massive datasets?

Next Generation Sequencing Signup and view all the answers

Which method is NOT typically used to detect proteins?

In situ hybridization Signup and view all the answers

In ex vivo gene therapy, how is the corrected DNA sequence reintroduced into the patient?

Inserted into cells removed from the body Signup and view all the answers

What is a major use of Bacterial Artificial Chromosomes (BACs)?

To clone large fragments of DNA Signup and view all the answers

What is the main purpose of using a probe in molecular biology?

To test for the presence of a specific DNA sequence Signup and view all the answers

Which of the following accurately describes the steps in PCR?

Denaturation, annealing, extension Signup and view all the answers

What does the term 'Tm' refer to in molecular biology?

Temperature for optimal primer binding Signup and view all the answers

Which cloning vector part is responsible for allowing the plasmid to replicate within a host organism?

Origin of replication Signup and view all the answers

What is a common challenge faced when transforming plant cells?

Cell walls that impede DNA uptake Signup and view all the answers

In the context of transformation, what does the term 'competent' refer to?

Preparation of cells to uptake DNA Signup and view all the answers

What is the primary advantage of using qPCR compared to traditional PCR?

It provides real-time data on DNA quantity during amplification. Signup and view all the answers

What is a significant advantage of using Arabidopsis thaliana in genetic studies?

Its genome is well known and it has unique antibiotics. Signup and view all the answers

What is a critical step that must be conducted after incubating cells with plasmids in transformation?

Heat shock cells to enhance plasmid uptake. Signup and view all the answers

What does site-directed mutagenesis aim to achieve?

To make precise modifications to specific DNA sequences Signup and view all the answers

Study Notes

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

Lower annealing temperature
Increase MgCl₂ concentration
Adjust genomic DNA amount

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

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Description

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.