Molecular Cloning Techniques Overview

Questions and Answers

Molecular cloning is used to construct recombinant ______ molecules.

DNA

The original PCR-restriction-ligation based cloning methods are still widely used for ______-scale investigations.

small

E. coli is typically the host organism used for maintaining ______ DNA.

foreign

A popular choice for cloning vectors when the DNA is relatively small is a ______-based cloning vector.

plasmid Signup and view all the answers

Cloning vectors include a selectable ______ to allow positive selection of transformants.

marker Signup and view all the answers

Molecular cloning has applications in the production of recombinant protein products such as ______.

hormones Signup and view all the answers

The final application of the cloning vector can involve protein ______ and purification.

expression Signup and view all the answers

Elements necessary for propagation and maintenance are required for plasmid ______ in E. coli.

replication Signup and view all the answers

A common technique to insert a PCR product into a cloning vector is to use restriction ______ to linearise the vector.

endonucleases Signup and view all the answers

PCR is widely used in molecular cloning to amplify the target ______ from the chosen organism.

DNA Signup and view all the answers

If the target DNA is prokaryotic, genomic DNA can be purified from ______ and directly used as a template.

cells Signup and view all the answers

To create complementary DNA, RNA can be converted using a retroviral enzyme called ______ transcriptase.

reverse Signup and view all the answers

In a typical PCR reaction of 30 cycles, each target sequence can theoretically generate 2^30 copies, which is over ______ billion.

1 Signup and view all the answers

DNA polymerase synthesises new complementary DNA strands in the ______ to 3' direction during the extension step.

5' Signup and view all the answers

Agarose gel electrophoresis is used to check the purity and ______ of the PCR product.

size Signup and view all the answers

To visualise DNA in the agarose gel, a nucleic acid ______ is used.

stain Signup and view all the answers

One commonly used fluorescent dye for staining DNA is ______ Safe.

SYBR® Signup and view all the answers

T4 DNA ligase is used to ligate the compatible sticky ends of the vector and ______ product.

PCR Signup and view all the answers

The annealing temperature during PCR should not be too ______ nor too high.

low Signup and view all the answers

Prior to ligation, the vector DNA may be treated with Antarctic ______ to prevent self-ligation.

phosphatase Signup and view all the answers

During the primer binding step, the temperature is reduced to ______ - 68°C.

48 Signup and view all the answers

Transformation allows the plasmid to be stably maintained and ______ for further experiments.

propagated Signup and view all the answers

Samples are loaded into the wells of a polyacrylamide gel under ______, and an electric field is applied across the gel to separate proteins.

buffer Signup and view all the answers

Proteins can either be separated in their native state (native PAGE) or in a ______ state by using a chemical such as sodium dodecyl sulphate.

denatured Signup and view all the answers

SDS is a negatively charged anionic ______ that denatures proteins by destroying their secondary, tertiary, and quaternary structure.

detergent Signup and view all the answers

In SDS-PAGE, the samples are heated to ______ °C for 5 – 10 min in a loading buffer containing SDS.

95-100 Signup and view all the answers

The reducing agent disrupts ______ bonds, helping to fully denature the proteins in the samples.

disulphide Signup and view all the answers

Coomassie Brilliant Blue R-250 is a negatively charged dye commonly used to ______ proteins in a gel following PAGE.

visualise Signup and view all the answers

After PAGE, the gel is placed onto a ______ membrane, which has a high affinity for proteins.

transfer Signup and view all the answers

The membrane is soaked for 1 – 2 hr in Tris-buffered saline containing 0.2% ______ to prevent non-specific binding of detection antibodies.

Tween-20 Signup and view all the answers

The primary antibody specifically binds to your ______ of interest during the Western blotting process.

protein Signup and view all the answers

The enzyme that generates smaller fragments which stimulate growth of the gland is called ______ Secretory Protease (AsP).

Adrenal Signup and view all the answers

Disruption of pituitary function can result in rapid atrophy of the ______.

adrenals Signup and view all the answers

The adrenal cortex produces steroid hormones including ______ and cortisol.

aldosterone Signup and view all the answers

AsP is produced as a zymogen, which is an ______ precursor.

inactive Signup and view all the answers

The activation peptide must be cleaved before the enzyme is ______.

active Signup and view all the answers

As a research scientist, you wish to investigate the substrate ______ and the enzyme kinetics of AsP.

specificity Signup and view all the answers

To achieve both aims you need a source of pure ______

enzyme Signup and view all the answers

You decide to generate recombinant protein by cloning the AsP ______ into a bacterial expression vector.

cDNA Signup and view all the answers

His tags allow rapid purification of the protein by affinity chromatography on an immobilized ______ column.

nickel Signup and view all the answers

The vector pET32A enables high expression levels in ______.

E.coli Signup and view all the answers

You will clone the rat adrenal secretory serine protease (AsP) cDNA into the pET32A vector to express a C-terminal ______ fusion protein.

HIS tagged Signup and view all the answers

Digestion of pET32a with NdeI and ______ will result in the release of two fragments.

XhoI Signup and view all the answers

Since AsP is a secreted protein, you need to create a construct that does not encode the secretory ______ sequence.

signal Signup and view all the answers

You need to include an AseI site in the forward primer and a ______ site in the reverse primer sequences.

XhoI Signup and view all the answers

The STOP codon is removed because you use the STOP codon present on the plasmid after the ______ tag coding sequence.

HIS Signup and view all the answers

You will need to check that the AsP cDNA is correctly expressed as a recombinant C-terminal HIS tag ______ protein.

fusion Signup and view all the answers

You will analyze the proteins using SDS-PAGE and ______ staining.

Coomassie Signup and view all the answers

Following IPTG induction, you should see a band at approximately ______ kDa in the induced culture.

29 Signup and view all the answers

You check the plasmid correctness by screening transformants using colony ______.

PCR Signup and view all the answers

The GeneRuler 1kb DNA Ladder mix contains ______ DNA fragments.

14 Signup and view all the answers

The Tris-acetate-EDTA running buffer consists of 0.04 M Tris-acetate; 0.001 M ______.

EDTA Signup and view all the answers

E.coli is not naturally competent, but there are two main methods used to transform E.coli in the laboratory by artificially inducing a temporary state of ______.

competence Signup and view all the answers

In chemical transformation, E.coli cells are treated with divalent cations, typically Ca2+ in the form of calcium ______.

chloride Signup and view all the answers

During chemical transformation, E.coli cells are subjected to a temporary heat shock at typically ______°C for 30 sec.

42 Signup and view all the answers

Electroporation punctures tiny holes in the cell membrane using a temporary ______ field.

electric Signup and view all the answers

Cells that do not take up a plasmid with a selectable marker cannot survive on selective ______.

media Signup and view all the answers

When an E.coli cell takes up a plasmid, the plasmid replicates within the cell ______.

autonomously Signup and view all the answers

Bacteria that have successfully taken up exogenous DNA are referred to as ______.

transformants Signup and view all the answers

Colony PCR can be used to screen transformants for the presence or absence of a foreign DNA ______.

insert Signup and view all the answers

It is possible to use colony PCR to check the ______ of the insert within the MCS.

orientation Signup and view all the answers

Plasmid DNA can be purified from a sample of each culture using ______ kits.

commercial Signup and view all the answers

If you have successfully cloned a gene of interest into an expression ______, you can transfer your construct into a host strain.

vector Signup and view all the answers

The presence of the inducer Isopropyl β-D-1-thiogalactopyranoside (IPTG) enables T7 polymerase to transcribe the recombinant ______.

gene Signup and view all the answers

A common method to detect recombinant proteins is through ______, which separates proteins based on their electrophoretic mobility.

polyacrylamide gel electrophoresis Signup and view all the answers

Acrylamide is highly toxic to humans in powder form, so pre-cast ______ gels are often used for safety reasons.

acrylamide Signup and view all the answers

Study Notes

Molecular Cloning

Molecular cloning involves constructing recombinant DNA molecules for replication in host organisms.
Traditional methods like PCR-restriction-ligation are used for small-scale cloning, while ligation-free methods are favored for larger projects such as cDNA libraries.
Applications include protein expression, recombinant protein production (e.g., insulin, growth hormone), vaccines, and genetically modified organisms.

Cloning Vectors

Integrating foreign DNA into suitable cloning vectors is essential for molecular cloning.
Factors in selecting a cloning vector include foreign DNA size, host organism (commonly E. coli), and intended application.
Plasmid vectors are often used for small foreign DNA, featuring:
- A cloning site (Multiple Cloning Site or MCS).
- A selectable marker (e.g., antibiotic resistance gene).
- Elements for propagation (e.g., origin of replication).
Vectors may also contain expression elements (promoters) and reporter genes (e.g., LacZα, GFP).

Polymerase Chain Reaction (PCR)

PCR is crucial for amplifying target DNA, especially from eukaryotic organisms where introns must be removed.
Complementary DNA (cDNA) is synthesized from RNA using reverse transcriptase to allow PCR amplification.
PCR typically uses a ready-to-use formulation called PCR Master Mix, consisting of DNA polymerase, dNTPs, MgCl2, and buffer.
A PCR cycle includes:
- Denaturation (94-98°C): Separates DNA strands.
- Annealing (48-68°C): Primers bind to target sequences.
- Extension (72°C): DNA polymerase synthesizes complementary DNA.
Theoretical exponential amplification can generate billions of DNA copies in standard cycles.
Agarose gel electrophoresis is used to check PCR product size and purity.

Restriction Endonucleases and Ligation

Restriction endonucleases linearize cloning vectors and create compatible ends for the insert DNA.
T4 DNA ligase joins sticky or blunt ends to form a new vector construct.
To prevent self-ligation, Antarctic phosphatase (AnP) dephosphorylates vector DNA ends.

Transformation of Competent E. coli

Transformation introduces the cloned construct into E. coli for maintenance and propagation.
E. coli is made competent via:
- Chemical transformation using CaCl2 and heat shock.
- Electroporation using electric fields to facilitate DNA uptake.
Transformants are selected on antibiotic-containing media.

Screening Transformants

Not all transformants carry the correct construct; screening is essential.
Colony PCR can identify the presence of foreign DNA inserts and their orientation.
Plasmid DNA is purified for confirmation through restriction mapping or sequencing.

Expression of Recombinant Proteins

Cloning a gene of interest into an expression vector allows protein expression in a suitable host strain.
For instance, the T7 expression system in E. coli utilizes T7 promoter and lac operon components.
Protein expression can be either constitutive or regulated based on the expression vector.

Detection of Recombinant Proteins

Proteins are detected using methods like SDS-PAGE and Western blotting.
SDS-PAGE involves denaturing proteins using SDS to ensure uniform charge-to-mass ratios for size-based separation.
After gel electrophoresis, proteins are transferred to membranes for detection with specific antibodies and visualized via various methods (e.g., chemiluminescence).

Adrenal Glands and AsP Research

Adrenal glands produce hormones such as adrenaline, noradrenaline, cortisol, and aldosterone, essential for stress response.
The adrenal cortex is regulated by anterior pituitary hormones, which also maintain gland size.
N-POMC is a peptide important for adrenal growth, cleaved at the gland to produce active fragments by Adrenal Secretory Protease (AsP).
AsP is a 279 amino acid serine protease produced as a zymogen with a 22 residue signal sequence and a 25 residue activation peptide.
The study aims to explore the substrate specificity and enzyme kinetics of AsP.### Recombinant Protein Production Strategy
Aim to produce pure AsP enzyme through recombinant protein expression.
Cloning AsP cDNA into a bacterial expression vector, avoiding purification from tissue samples.

Fusion Protein and His Tag

Creation of a fusion protein by adding a His tag to the C-terminal of AsP.
His tags facilitate detection with commercial antibodies and allow for efficient purification via affinity chromatography on immobilized nickel columns.

Expression Vector Choice

Selection of pET32A vector for high expression levels in E. coli.
pET32A also includes a thioredoxin tag for N-terminal fusion, though the construct designed excludes this.

Cloning Strategy

Restriction enzyme digestion and ligation are used to clone AsP cDNA into pET32A.
NdeI and XhoI chosen for vector linearization, enabling cloning while maintaining the His tag sequence.

Construct Design for E. coli

AsP gene will be expressed without the secretory signal sequence; necessary ATG (start codon) added at the N-terminal.
Confirm no XhoI sites in AsP cDNA, with one NdeI site located between positions 531-536.

Alternative Restriction Enzyme

AseI recognized for producing compatible ends with NdeI, ensuring no AseI sites present within the AsP sequence.

Primer Design

Forward primer includes AseI site, reverse primer includes XhoI site.
Reverse primer lacks the AsP stop codon for correct termination post-His tag translation.

Verification Process

Colony PCR screening for correctly cloned plasmid using designed primers.
Confirmation through restriction enzyme digestion to verify proper insertion of AsP cDNA.

Protein Induction and Visualization

Transfer recombinant plasmid to E. coli BL21(DE3) cells and induce with IPTG.
Separation and visualization of proteins via SDS-PAGE, expecting a band at approximately 29 kDa for successfully expressed AsP fusion protein.

Electrophoresis Buffers and Reagents

TAE running buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0) used for DNA gel electrophoresis.
1x SDS running buffer contains 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3.
InstantBlue® Coomassie Protein Stain employed for visualizing proteins in SDS-PAGE.

Protein Standards

Bio Rad Precision Plus Protein Dual Colour Standard used as a reference for protein molecular weight estimation.

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Description

Explore the fundamental techniques of molecular cloning, a crucial process in genetic engineering. This quiz covers various methods used to construct recombinant DNA and the advancements made in the field. Test your understanding of both traditional and modern cloning techniques.