Recombinant DNA Technology Overview

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Questions and Answers

What is the purpose of primers in a PCR reaction?

To bind to the target DNA sequence (correct)

To provide DNA polymerase

To supply necessary nucleotides

To catalyze the DNA synthesis

What is the role of DNA ligase in the process of recombinant DNA technology?

To join two DNA fragments together (correct)

To introduce recombinant DNA into host cells

To amplify the DNA

To cut the DNA into fragments

Which method is typically NOT used to make bacterial cells competent for DNA transformation?

Electroporation

Thermal shock

Magnetic field exposure (correct)

Heat shock

Why is a marker gene used in the selection of transformed cells?

To differentiate between recombinant and non-recombinant cells (C) Signup and view all the answers

What is the result of successful transformation in recombinant DNA technology?

Introduction of recombinant DNA into a recipient host cell (B) Signup and view all the answers

What is Recombinant DNA commonly referred to as?

Chimeric DNA (C) Signup and view all the answers

Which process is NOT a method used to create Recombinant DNA?

Translocation (D) Signup and view all the answers

What is the purpose of the selectable marker in the inserted DNA?

To identify recombinant molecules (C) Signup and view all the answers

Which scientist was the first to insert recombined genes into bacterial cells?

Stanley Cohen (B) Signup and view all the answers

What is the function of a restriction enzyme in the Recombinant DNA process?

To cut DNA at specific sites (C) Signup and view all the answers

In microinjection, how is DNA introduced into the host cell?

It is injected into the nucleus (A) Signup and view all the answers

Which of the following describes Biolistic transformation?

Bombarding host cells with micro-projectiles (A) Signup and view all the answers

What is a common characteristic of a host cell in the Recombinant DNA process?

It must be resistant to antibiotics (C) Signup and view all the answers

What was the primary contribution of Hamilton O. Smith in the development of Type II restriction enzymes?

Purifying Type II restriction enzymes (B) Signup and view all the answers

Which process involves adding a methyl group to DNA and can influence gene expression?

DNA methylation (B) Signup and view all the answers

What marks the first step in recombinant DNA technology?

Isolation of genetic material (B) Signup and view all the answers

What role do restriction enzymes play in recombinant DNA technology?

They cut DNA at specific locations (C) Signup and view all the answers

In the context of agarose gel electrophoresis, what characteristic of DNA allows it to move towards the positive electrode?

Negatively charged nature of DNA (A) Signup and view all the answers

What is the purpose of polymerase chain reaction (PCR) in recombinant DNA technology?

To amplify DNA sequences (D) Signup and view all the answers

Which scientists were the first to successfully insert recombined genes into bacterial cells?

Stanley N. Cohen and Herbert W. Boyer (D) Signup and view all the answers

What is the final step involved in the process of isolating DNA in recombinant DNA technology?

Addition of ethanol (D) Signup and view all the answers

Study Notes

Recombinant DNA Technology (Background)

Recombinant DNA is the general name for combining a piece of one DNA strand with another DNA strand
It's also sometimes called a "chimera"
The most common method combines DNA from two different organisms
There are three different methods for making recombinant DNA: Transformation, Phage Introduction, and Non-Bacterial Transformation

Transformation

Select a piece of DNA to be inserted into a vector
Cut the selected DNA with a restriction enzyme
Ligate the DNA insert into the vector with DNA ligase
The inserted DNA contains a selectable marker for identifying recombinant molecules
An antibiotic marker is often used; host cells without a vector die when exposed to the antibiotic; host cells with the vector live because they are resistant
The vector is inserted into a host cell, which is transformation
E. coli is an example of a host cell; host cells must be specially prepared

Non-Bacterial Transformation

In microinjection, DNA is directly injected into the nucleus of the cell being transformed
In biolistic, host cells are bombarded with high-velocity micro-projectiles (gold or tungsten particles coated with DNA)

Historical Background

Restriction enzymes were discovered in 1968 by Werner Arber.
Type II restriction enzymes were discovered in 1969 by Hamilton O. Smith.
Daniel Nathans advanced the technique of DNA recombination, showing type II enzymes could be useful in genetic studies (1970-71)
Paul Berg developed methods for splitting DNA molecules and attaching other DNA segments (1973)
Stanley N. Cohen and Herbert W. Boyer first inserted recombined genes into bacterial cells in 1973

DNA Methylation

Methylation adds a methyl group to a DNA base
This can affect gene expression and health

Steps in Recombinant DNA Technology

Isolation of Genetic Material

The first step is isolating the desired DNA from other macromolecules
Since DNA exists within the cell membrane with other macromolecules (RNA, polysaccharides, proteins, and lipids), it must be separated and purified
This often involves enzymes like lysozyme, cellulose, chitin, ribonuclease, and proteases
Ethanol is used to precipitate the DNA

Restriction Enzyme Digestion

Restriction enzymes act as molecular ‘scissors’ cutting DNA at specific locations
They incubate the purified DNA with a selected restriction enzyme at optimal conditions
The technique 'Agarose Gel Electrophoresis' is used to separate DNA fragments based on size after the digestion

Amplification Using PCR

Polymerase Chain Reaction (PCR) is a method for making multiple copies of a DNA sequence
It amplifies a single copy or a few copies of DNA into thousands or millions
Key components of PCR include template (DNA to be amplified), primers (small oligonucleotides complementary to a region of the DNA), enzyme (DNA polymerase), and nucleotides

Ligation of DNA Molecule

The purified DNA and the vector of interest are cut using the same restriction enzyme
The ends are joined together using DNA ligase, forming a hybrid DNA molecule.
Hybrid DNA molecule is also called a recombinant DNA molecule
The technology behind this process is called recombinant DNA technology

Insertion of Recombinant DNA into Host

Recombinant DNA is introduced into a recipient host cell (often a bacterial cell) This process is called transformation
Bacterial cells are treated to become competent to take up foreign DNA, such as using thermal shock, Ca++ ion treatment, electroporation, or other methods.

Isolation of Recombinant Cells

The transformation process creates a mixed population of transformed and non-transformed host cells
Selection techniques filter out only the transformed host cells
Marker genes (e.g., in plasmid vectors like PBR322, including Ampicillin and Tetracycline resistant genes) are used to distinguish recombinant cells from non-recombinant cells

Application of Recombinant DNA Technology (example)

Scientific research
Medicine and pharmacology industry
Pest or insecticide-resistant plants
Bacteria used to clean oil or toxic spills
Food industry

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Description

This quiz explores the fundamental concepts of recombinant DNA technology, including the techniques used to create recombinant DNA like transformation and non-bacterial transformation. Test your understanding of the methods, vectors, and host cells involved in this crucial area of genetic engineering.