Recombinant DNA Overview

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Questions and Answers

What is recombinant DNA (rDNA)?

Recombinant DNA (rDNA) is DNA that has been formed by laboratory methods of genetic recombination. It creates new DNA sequences by combining genetic material from multiple sources.

RDNA is only possible in organisms that share the same chemical structure.

False

What is rDNA sometimes called and why?

rDNA is sometimes called chimeric DNA because it can be made of material from two different species.

Recombinant DNA technology uses palindromic sequences which leads to the production of sticky and blunt ends.

True Signup and view all the answers

What is recombinant DNA?

Recombinant DNA is DNA that has been artificially formed by combining constituents from different organisms. Signup and view all the answers

What does recombinant DNA technology allow us to do?

Recombinant DNA technology allows us to isolate and clone single copy of a gene or a DNA segment into an indefinite number of identical copies. Signup and view all the answers

What is the first step in making recombinant DNA?

The first step is to isolate donor and vector DNA. Signup and view all the answers

What is a commonly used vector in recombinant DNA technology?

Bacterial plasmids are commonly used vectors. Signup and view all the answers

What method can be used to extract plasmids DNA?

Ultracentrifugation can be used to extract plasmids DNA. Signup and view all the answers

What is another method for extracting plasmids DNA?

Another method is alkaline lysis. Signup and view all the answers

What do restriction enzymes do in rDNA technology?

Restriction enzymes cut DNA molecules at specific recognition sequences, resulting in linear molecules with sticky or blunt ends. Signup and view all the answers

What is a vector in rDNA technology?

A vector is a DNA molecule capable of multiplying inside a host cell. Signup and view all the answers

How do restriction enzymes function in the process of creating rDNA?

They function as scissors, cutting the DNA molecule, while ligase enzyme joins the vector DNA with the gene of interest. Signup and view all the answers

How are plasmids introduced into E.coli during rDNA technology?

Plasmids are added to a flask containing a culture of E.coli. Calcium ions and a brief heat shock are used to make the cell membrane permeable to DNA. Signup and view all the answers

How are phage vectors introduced into bacterial cells?

Phage vectors are introduced by infection of bacterial lawns growing on an agar plate. Signup and view all the answers

How are plasmids purified before cutting with a restriction enzyme?

Bacterial cells are broken open and their chromosomal DNA is centrifuged down. Signup and view all the answers

What happens after restriction fragments from donor DNA are mixed with plasmid DNA?

The fragments join by their sticky ends, first through hydrogen bonds, and then the sugar phosphate backbone is joined by an enzyme called DNA ligase. Signup and view all the answers

Describe the process of producing insulin using recombinant DNA technology.

The DNA for insulin is isolated, and a plasmid made of DNA is removed from a bacterial cell. A restriction enzyme cuts the plasmid DNA open, leaving sticky ends. The insulin gene with complementary sticky ends is added, and DNA ligase joins the plasmid DNA and the insulin DNA. The modified plasmid is inserted back into the bacterium, which then produces copies of the plasmid and makes human insulin using the gene. Signup and view all the answers

What are some of the applications of recombinant DNA technology?

Recombinant DNA technology is used in the preparation of gene maps. It can reveal details of various infections and diseases. It aids in finding out the complete nucleotide sequence of an organisms genome. Additionally, it can detect cytogenetic abnormalities like Down's syndrome, prevent inherited genetic disorders like phenylketonuria, and shed light on biological processes like growth, differentiation, and ageing. It is also used in gene therapy, where defective genes are replaced with normal genes to treat genetic diseases. Signup and view all the answers

What are some of the potential negative features of recombinant DNA technology?

Recombinant DNA technology has potential negative features including extensive erosion and genetic destruction of plant germplasm, ecological imbalance, the potential for the production of dangerous toxic chemicals, and the possibility of generating highly lethal microbes. Signup and view all the answers

Study Notes

Recombinant DNA (rDNA) Definition

rDNA are DNA molecules formed through laboratory methods of genetic recombination
This involves combining genetic material from different sources
rDNA creation bypasses natural genetic combinations
rDNA is possible because all organisms' DNA shares a similar chemical structure
Variations lie within the nucleotide sequence

Recombinant DNA (rDNA) Introduction

Recombinant DNA is a general term for DNA created through combining at least two DNA strands
Recombinant DNA molecules are also known as chimeric DNA
This name is for the ability to combine material from different species, similar to mythical chimeras
rDNA technology uses palindromic sequences to produce sticky and blunt ends of DNA

Recombinant DNA Technology Overview

Recombinant DNA technology allows isolation and cloning of specific gene or DNA segments
Creates numerous identical copies
New genetic combinations are introduced (rDNA) into host cells
Host cells propagate and multiply the new genetic material, thereby producing identical copies
The process of this methodology is called Recombinant DNA technology

Obtaining Recombinant DNA (rDNA) - Steps

Step 1: Isolate the DNA segment to be cloned (the insert)
Step 2: Cut DNA using specific enzymes (restriction enzymes)
Step 3: Join the DNA segments together using DNA ligase
Step 4: Insert the resulting rDNA into a host carrier (a vector)
Step 5: Allow the DNA to replicate and create identical copies in the host cell
Step 6: Transfer the modified DNA to an appropriate host cell
Step 7: Select host cells carrying the rDNA molecule using a marker
Step 8: Permit rDNA replication to generate a genetically uniform clone collection

rDNA Isolation

The first step is isolating donor and vector DNA
Vector DNA isolation method depends on the vector's nature (e.g., bacterial plasmids)
Plasmids must be purified from the bacterial genome

rDNA Isolation - Techniques

Ultracentrifugation: Separates plasmid DNA from bacterial genomic DNA based on density differences
Alkaline Lysis: Denatures genomic DNA at a given alkaline pH, while leaving plasmids intact. Neutralization precipitates the genomic DNA, leaving plasmids in solution.

Cutting DNA - Restriction Enzymes

Restriction enzymes (e.g., EcoRI) cut DNA at specific sequences creating sticky ends
This is used to introduce foreign DNA fragments into a vector

Insertion

Vector: A DNA molecule that replicates inside a host cell and integrates the desired gene of interest.
Restriction enzymes act as genetic scissors
Ligase enzyme joins DNA fragments together

Introducing Vector DNA into Host Cells

Plasmid vectors are added into the host cell culture (e.g., E. coli)
Calcium ions and heat shock create temporary holes in the host cell membrane
This allows plasmid DNA to enter

Phage Vectors

Cloning technique involving bacterial infection utilizing phage vectors
Phage viruses introduce DNA to a bacterial host
Growing viruses is more difficult than bacteria or fungi

Placing the Gene in the Vector

Plasmid DNA are small, making separation by size easy from bacterial chromosomes
Chromosomal DNA is removed
The plasmid DNA is then purified

Joining DNA

Restriction fragments from Donor DNA are mixed with plasmid DNA.
Sticky ends allow for initial hydrogen bond attraction.
The sugar-phosphate backbone is joined by DNA ligase.

Recombinant DNA Technology Applications

Genetic mapping: Mapping genes on DNA.
Infectious disease/genetic disorder research: Understanding diseases and infections
Genome sequencing: Determining an organism's complete DNA sequence
Genetic abnormalities detection: Finding chromosomal disorders, such as Down syndrome
Disease prevention: Preventing/treating inherited disorders (e.g., sickle cell anemia, phenylketonuria)
Cellular processes research: Investigating molecular mechanisms underlying biological processes including growth, differentiation, and aging
Gene therapy: Introducing functional genes to correct defective genes (e.g., sickle cell anemia)
GMO production: Creating organisms (GMOs or transgenic organisms) with desired traits/features

Negative Aspects of Recombinant DNA Technology

Erosion of genetic diversity in plants,
Ecological damage,
Creation of dangerous toxins/microbes, and
Potential misuse in biological warfare.

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Description

This quiz covers the basics of recombinant DNA (rDNA), including its definition, creation methods, and applications in genetic technology. Explore how rDNA enables the combination of different genetic materials and the significance of this process in modern biology.