Molecular Cloning Basics

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Questions and Answers

What is the first step in the molecular cloning process?

Transfer recombinant DNA into a host

Analyze mutant vs WT genes

Cut and paste vector to the DNA fragment of interest

Make recombinant DNA via attaching vector and insert (correct)

Which component of a vector ensures its replication within the host?

Protein-coding region

Origin of replication (correct)

Multiple cloning site

Selectable marker

What is one of the reasons for using molecular cloning techniques?

To increase the number of cells that do not contain recombinant DNA

To prevent mutations from occurring in the DNA

To isolate and analyze specific regions of DNA (correct)

To delete sections of DNA from the genome

Which feature allows you to select host cells that carry recombinant DNA?

Selectable marker (B) Signup and view all the answers

What limitation do plasmids have as vectors in molecular cloning?

Size of the insert (D) Signup and view all the answers

What is the purpose of the multiple cloning site in a vector?

It provides restriction enzyme sites for inserting genes (B) Signup and view all the answers

What is molecular cloning primarily used for?

To analyze DNA sequences and gene expression (C) Signup and view all the answers

What type of DNA is often used in molecular cloning as a vector?

Plasmid DNA (A) Signup and view all the answers

What is a major advantage of using immunoprecipitation?

It provides better results with native protein. (D) Signup and view all the answers

What is a necessary step before conducting chromatin immunoprecipitation?

Crosslinking DNA-protein interactions. (C) Signup and view all the answers

In the yeast two-hybrid system, what happens when the bait and prey proteins interact?

The reporter gene is transcribed. (B) Signup and view all the answers

What is a common challenge faced in the use of immunoprecipitation?

Difficulty in making effective antibodies. (B) Signup and view all the answers

What is the role of affinity ligands in protein purification?

To hold the fusion protein for separation. (C) Signup and view all the answers

What is the primary function of T4 DNA polymerase in relation to overhangs?

It removes 3' overhangs. (B), It fills in 5' overhangs. (C) Signup and view all the answers

Which method involves altering E. coli to allow DNA uptake?

Electroporation (A) Signup and view all the answers

What characteristic does PCR exploit for exponential DNA amplification?

Template-dependent synthesis (D) Signup and view all the answers

What feature is necessary for primers used in PCR?

Minimum of 17 bp (A) Signup and view all the answers

What does the melting temperature (Tm) of a primer indicate?

Temperature at which a primer will dissociate from template DNA (A) Signup and view all the answers

Why is blunt-end cloning less commonly used?

It is not efficient and lacks directionality. (C) Signup and view all the answers

What do PCR primers need to provide for DNA polymerase to function effectively?

A free 3’ hydroxyl group (D) Signup and view all the answers

Which enzyme is commonly associated with reverse transcription in RT-PCR?

Reverse transcriptase (C) Signup and view all the answers

What is an important factor to ensure when calculating the annealing temperature (Ta) for PCR?

It should be about 5°C lower than the Tm. (D) Signup and view all the answers

Which property of DNA polymerases is particularly important during PCR?

Heat stability and processivity (C) Signup and view all the answers

What is a significant advantage of incorporating restriction sites into PCR primers?

It creates sticky ends after digesting the PCR product. (C) Signup and view all the answers

What is achieved during the first strand synthesis in RT-PCR?

cDNA is synthesized from RNA using reverse transcriptase. (C) Signup and view all the answers

Which of the following statements reflects a challenge in molecular cloning?

Not all colonies will contain vectors. (C) Signup and view all the answers

How does Taq polymerase affect the ends of its PCR products?

Adds a 3' A overhang (C) Signup and view all the answers

What is the primary purpose of qPCR during the PCR process?

To measure the amount of DNA during the exponential phase of PCR. (A) Signup and view all the answers

Which component is essential for the synthesis of DNA during PCR?

dNTPs (B) Signup and view all the answers

What does a lower Ct value in qPCR indicate?

There is more template DNA present initially. (A) Signup and view all the answers

How does the process of Sanger sequencing determine the identity of nucleotides in a DNA sequence?

By incorporating ddNTPs, which terminate DNA synthesis. (A) Signup and view all the answers

What is the role of housekeeping genes in ΔΔCt analysis?

As reference samples to normalize data. (B) Signup and view all the answers

Which statement best differentiates RT-PCR from qPCR?

RT-PCR can be performed without quantitatively measuring the product. (C) Signup and view all the answers

What method is commonly used to visualize DNA after electrophoresis?

Ethidium Bromide staining (B) Signup and view all the answers

Why is it important to perform a mini prep in molecular cloning?

To isolate plasmid DNA from bacteria. (C) Signup and view all the answers

How do fluorescent probes enhance the capabilities of qPCR?

By allowing for multiplexing of different targets. (A) Signup and view all the answers

What is the function of using a DNA ladder in electrophoresis?

To estimate the size of separated DNA fragments. (D) Signup and view all the answers

What is the significance of the ΔCt value in qPCR analysis?

It provides a measure of relative template amounts between samples. (C) Signup and view all the answers

What type of DNA analysis uses primers that attach to both the vector and the gene of interest?

PCR (B) Signup and view all the answers

What is a potential use of PCR outside of cloning processes?

Genetic fingerprinting (A) Signup and view all the answers

In DNA sequencing, what is the purpose of modified dNTPs?

To terminate DNA strand synthesis. (C) Signup and view all the answers

What is the primary role of restriction enzymes in DNA cloning?

To cut DNA at specific recognition sites. (D) Signup and view all the answers

How do Type II restriction enzymes differ from other types?

They cut DNA at specific positions related to recognition sites. (D) Signup and view all the answers

What happens when DNA is cut by two different restriction enzymes?

The ends will be incompatible and unable to stick together. (A) Signup and view all the answers

What is the function of DNA ligase in the cloning process?

To form phosphodiester bonds between DNA fragments. (D) Signup and view all the answers

What role does temperature play in the efficiency of the ligation reaction?

Lower temperatures stabilize hydrogen bonds for sticky ends. (B) Signup and view all the answers

What is the importance of having 5' phosphates on DNA for ligation?

5' phosphates enable the formation of phosphodiester bonds. (A) Signup and view all the answers

Why might a phosphatase treatment be used on a vector during cloning?

To prevent the vector from ligating to itself. (D) Signup and view all the answers

Which of the following statements about restriction enzymes is true?

Restriction enzymes recognize specific, short DNA sequences. (B) Signup and view all the answers

What occurs during ligation at the molecular level?

A phosphodiester bond is formed between two DNA strands. (D) Signup and view all the answers

What is a major limitation of blunt-ended ligation compared to sticky-ended ligation?

Blunt-ended ligation is less efficient due to lack of temporary hydrogen-bonding. (D) Signup and view all the answers

What is the significance of nucleases in relation to phage DNA in bacterial hosts?

Nucleases degrade unmodified phage DNA in foreign hosts. (C) Signup and view all the answers

What must be ensured to successfully ligate DNA fragments cut with different restriction enzymes?

Both fragments should have the same sticky ends. (C) Signup and view all the answers

What occurs when a DNA sequence is modified by methylation?

The sequence is protected from degradation by restriction enzymes. (B) Signup and view all the answers

How do mismatch nucleotides affect the activity of restriction enzymes?

They can eliminate the enzyme's ability to cut at the recognition site. (C) Signup and view all the answers

What is the main function of dideoxynucleotides (ddNTPs) in DNA sequencing?

To compete with dNTPs during DNA synthesis (C) Signup and view all the answers

What is the primary method used to measure transcriptional activity of multiple genes simultaneously?

Microarrays (B) Signup and view all the answers

Which of the following methods can be used to visualize RNA localization in cells?

Fluorescent in situ hybridization (FISH) (D) Signup and view all the answers

What is the central principle of the northern blot technique?

Detection of specific RNA sequences (A) Signup and view all the answers

Which reporter gene is commonly used to visualize promoter activity?

Luciferase (C) Signup and view all the answers

What is the purpose of using secondary antibodies in protein detection methods?

To label primary antibodies for visualization (B) Signup and view all the answers

Which method is primarily used to determine protein size during analysis?

Gel electrophoresis (B) Signup and view all the answers

What role do oligonucleotides play in microarray experiments?

They serve as probes attached to the chip (C) Signup and view all the answers

In the context of protein-protein interactions, what is the purpose of a pull-down assay?

To purify and identify interacting proteins (A) Signup and view all the answers

What is the main disadvantage of using northern blotting for gene expression analysis?

It cannot analyze multiple genes at once (A) Signup and view all the answers

What influence does the presence of a fluorescent fusion protein have on protein localization studies?

It provides real-time imaging of protein dynamics (B) Signup and view all the answers

Which component is essential for the successful operation of a PCR reaction?

DNA polymerase (D) Signup and view all the answers

What is one of the primary uses of chromatin immunoprecipitation (ChIP)?

Analyzing protein interactions with DNA (A) Signup and view all the answers

What characteristic differentiates RNA from DNA in the context of molecular biology?

RNA is usually single-stranded (A) Signup and view all the answers

Study Notes

Molecular Cloning Overview

Molecular cloning is a set of methods in molecular biology used to make recombinant DNA molecules.
Recombinant DNA combines DNA sequences from different sources (organisms or synthetic).
Cloning is used to isolate specific DNA regions (genes, coding sequences, or promoters).
Downstream applications include gene sequencing, analyzing mutations, creating mutations, expressing and purifying proteins.

Steps in Molecular Cloning

Step 1: Create recombinant DNA by joining a vector and insert (target DNA).
Step 2: Introduce recombinant DNA into a host cell, grow, and clone the recombinant DNA in the host.
Step 3: The vector is cut and pasted with the DNA fragment of interest.

Cloning Vectors

A vector is DNA capable of entering and replicating in a host cell.
Commonly used vector: plasmid (circular DNA outside bacterial chromosomes).
Essential vector features:
- Origin of replication: Enables independent replication in the host, crucial for vector maintenance. High or low copy numbers refer to the number of plasmid copies per cell.
- Selectable marker: Allows identification of host cells containing the vector (e.g., antibiotic resistance).
- Multiple Cloning Site (MCS): Contains multiple restriction enzyme recognition sites allowing for insertion of foreign DNA.

Types of Vectors

Plasmids, circular DNA outside a cell's chromosome.
Other vector types exist but are less frequently used in basic cloning.

Cloning DNA into a Vector

Cutting DNA: Use restriction enzymes to cut DNA at specific locations.
Joining DNA: Use DNA ligase to join DNA fragments together.
Preparing DNA Insert: Cut, amplify, or purify DNA based on its starting form (e.g. PCR product or restriction fragment).
Modifying DNA Ends: Alter DNA ends to ensure compatibility with the vector.

Restriction Enzymes

Many types/hundreds in use.
Type II: Most common, cut DNA at precise locations near or within their recognition site, generating sticky or blunt ends. Typically homodimers.
Recognition sites are usually palindromes (read the same forwards and backwards).
Type I and III: Cut randomly far from the recognition sequence.
Type IV: Cut modified DNA.

Sticky vs. Blunt Ends

Sticky ends are complementary overhanging sequences that can base pair.
Blunt ends have no overhangs.
Specific enzymes generate specific sticky end overhangs, which will only be compatible if digested with the same type of enzyme.
Blunt ends are less efficient for ligation (no sticky ends)

Ligation Reactions (Joining DNA)

DNA ligase catalyzes the formation of phosphodiester bonds between DNA fragments.
Lower temperatures generally enhance ligation efficiency.

DNA Ligase Mechanism

DNA ligase uses ATP to form an AMP intermediate.
The AMP is transferred to the 5'-phosphate, and then attacked by the 3'-OH forming the phosphodiester bond.

Potential Cloning Issues

Vector self-ligation: Potential problem if the vector can ligate to itself instead of the insert. Addressing by using phosphatase (CIP)- treatment removes 5’phosphate from the vector.
Incorrect orientation of the insert can be solved by using more than one restriction enzyme for both the ends of the gene being cloned.

Modifying DNA Ends

Adding/Removing 5' phosphate: Phosphatase removes the 5' phosphate. Needed for directional cloning.
Removing overhangs: T4 DNA polymerase or DNA polymerase I used for filling in 5' overhangs or removing 3' overhangs. Mung bean nuclease removes 5' overhangs.

Transformation

Electroporation: Using a high-voltage pulse.
Chemical transformation (heat shock).

Selection and Amplification

Identify which bacterial colonies contain the desired recombinant plasmids using selectable markers.

PCR (Polymerase Chain Reaction)

PCR is an in vitro method to amplify a specific DNA sequence.
PCR methods include RT-PCR and quantitative PCR (qPCR).
PCR uses specific primers that flank the target DNA to amplify it.

DNA Polymerases in PCR

Thermostable polymerases (e.g. Taq, Pfu) maintain activity under high temperatures.
Choose polymerase based on desired accuracy, speed, or processivity qualities.

PCR Primers

Primers are short, single-stranded DNA sequences complementary to the DNA region to be amplified. At least 17 base pairs may be needed, with 20 typically used.
Primers need specific melting temperatures that are generally around 60-64C.
Calculate melting temperature using programs or older calculations of 2 or 4 degrees for each base.

PCR Variants

RT-PCR: Amplifies cDNA from RNA (reverse transcription needed first).
qPCR: Measures DNA amplification in real time using fluorescent dyes. Useful quantifying DNA.

DNA Sequencing

Sanger sequencing (chain termination method) determines DNA sequences.
Automated sequencing uses fluorescent dyes, capillary electrophoresis, and computer analysis.

Gene Expression Analysis Methods

PCR-based methods: RT-PCR for measuring RNA levels.
Hybridisation-based methods: Northern blots (single gene), microarrays (multiple genes), in situ hybridisation (localisation).
Reporter genes: Used to measure promoter activity in live organisms or cells.
Antibody-based methods: Western blots (proteins) and immunofluorescence (proteins).

Fluorescent Protein Fusion Systems

Fusion of protein of interest with fluorescent proteins allows visualization of molecules and interactions in situ/live organisms.

Protein-Protein Interactions

Various techniques to identify interacting protein partners: pull-down assays, immunoprecipitation, yeast two-hybrid, and chromatin immunoprecipitation (ChIP).

Other Useful Points

Restriction mapping is a technique for determining the positions of restriction enzyme recognition sites on a DNA molecule to create a map.
Electrophoresis is used to separate DNA/RNA/protein based on size.

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Test your understanding of the fundamental concepts of molecular cloning. This quiz covers essential components such as vectors, their features, and applications in genetic research. Ideal for students and professionals looking to brush up on their molecular biology knowledge.