Recombinant DNA Technology and Gene Cloning

Recombinant DNA Technology

• Recombinant DNA is a piece of DNA created by inserting a DNA fragment from one organism into the replicating DNA of another.
• It involves combining at least two DNA molecules.
• Recombinant DNA (rDNA) molecules are formed by laboratory methods of genetic recombination, such as molecular cloning, to combine genetic material from multiple sources.
• These DNA sequences are not naturally found in a genome, created via genetic engineering.
• rDNA sequences can come from any organism.
• Recombinant DNA technology and synthetic DNA allow the creation of any DNA sequence and its introduction into living organisms.
• Recombinant DNA technology and genetic engineering center around gene cloning.

Gene Cloning

• Introducing a foreign DNA molecule into a replicating cell permits cloning or amplification of that DNA.
• This produces many identical copies of the DNA of interest.
• PCR is an alternative to gene cloning.

Basic Steps of Gene Cloning

• Step 1: Isolate the DNA fragment containing the gene to be cloned and insert it into a circular DNA molecule, called a vector, to make recombinant DNA.
• Step 2: The vector transports the gene into a host cell (usually a bacterium).
• Step 3: Inside the host cell, the vector multiplies, creating numerous copies of the gene and itself.
• Step 4: When the host cell divides, the recombinant DNA is passed on to the offspring and replicates further.
• Step 5: After many cell divisions, a colony or clone of identical host cells is developed, and each cell in the clone will contain numerous copies of the recombinant DNA. The gene carried by the recombinant molecule is now said to be cloned.

Vectors for Gene Cloning

• A vector is a DNA molecule to which the fragment of DNA to be cloned is attached.
• Common vectors include plasmids and viruses.

Essential Properties of a Vector

• Vectors must be able to replicate within the host cells.
• They must have the ability for autonomous replication in the host cell (many copies).
• Ideally, vectors should be relatively small (less than 10 kb) to prevent degradation during purification.
• Vectors should contain at least one specific nucleotide sequence recognizable by restriction endonucleases.
• It must have at least one gene that controls the selection of the vector (like antibiotic resistance genes).

Plasmids

• Plasmids are circular, double-stranded DNA molecules that exist independently within bacterial cells.
• Plasmids often contain one or more genes that are responsible for specific characteristics of the host bacterium. (Example: antibiotic resistance genes).
• Plasmids often have an origin of replication, enabling them to replicate independently of the host chromosome.
• In the lab, antibiotic resistance can often be used as a marker to identify which bacteria contain the specific plasmid.

Size and Copy Number of Plasmids

• The ideal size for a plasmid is less than 10 kb.
• High copy number of the plasmids is favored (high number of plasmid molecules per bacterial cell). A high copy number is desired to obtain large quantities of the recombinant DNA.
• Low copy number plasmids might be preferable in specific conditions (e.g., toxin production from the cloned gene).

Bacteriophages

• Bacteriophages, or phages, are viruses that specifically infect bacteria.
• They are simple in structure, consisting of a DNA molecule embedded within a protein coat (capsid).

Bacteriophage Lambda as a Cloning Vector

• Bacteriophage lambda can be modified to serve as a vector for cloning to carry large DNA fragments (10–23 kb).

The General Pattern of Infection of a Bacterial Cell by a Bacteriophage

• The phage attaches to the bacterium and injects its DNA.
• The phage DNA molecule is replicated.
• Capsid components are synthesized, phage particles are assembled, and released from the host cell.

Other Vectors

• Naturally occurring viruses that infect mammalian cells, like retroviruses, can be used as vectors to clone large DNA fragments.
• Artificial constructs, such as cosmids, provide alternative cloning vectors.
• Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are other vectors that can accommodate large DNA fragments.

Cloning Vectors and Their Insert Capacities

• Tables present various cloning vector systems and their respective insert capacities, outlining compatibility with host cells and DNA fragment sizes.

Enzymes for Cutting DNA - Restriction Endonucleases

• DNA molecules need to be precisely cut.
• Restriction enzymes cut DNA molecules at specific positions.
• Each particular restriction enzyme only recognizes one specific sequence.
• Enzymes cut DNA at a particular nucleotide sequences, termed the "recognition sequence"
• Some recognition sequences are palindromes.

Type II Restriction Endonucleases

• Type II restriction endonucleases cut DNA at specific nucleotide sequences.
• Each enzyme has a specific recognition sequence at which it cuts a DNA molecule, but nowhere else.

A Palindrome

• A palindrome reads the same forward and backward between the 5' and 3' directions

Type II Restriction Endonucleases Examples

• Restriction enzymes recognize specific hexanucleotide sequences, such as CGATCG (by PvuII).
• Some recognize 4, 5, 8, or more nucleotides and recognize a family of related sequences.
• Specific examples for recognition sequences (and enzymes) are shown in a table.

Blunt Ends and Sticky Ends

• Certain restriction enzymes make a straight cut in the DNA (blunt ends), while others generate staggered cuts, creating overhangs (sticky ends).
• Blunt ends are simpler to work with, but sticky ends facilitate ligation.

Formation of Recombinant DNA from Restriction Fragments with "Sticky" Ends

• "Sticky" ends (overhangs) facilitate the ligation process, as complementary bases pair to join fragments.

Ligation - Joining DNA Molecules Together

• Ligation involves joining DNA fragments using DNA ligase.
• DNA ligase catalyzes this reaction, joining individual DNA molecules or the ends of the same molecule.
• The reaction joins phosphodiester bonds.

Ligation of Blunt-Ended and Sticky-Ended Molecules

• Ligation process diagrams represent both blunt-ended and sticky-ended molecule ligations, showcasing differences clearly.

Sticky Ends Increase the Efficiency of Ligation

• Sticky ends, with their complementary sequences, increase the efficiency of ligation, as they allow the fragments to align correctly. Ligation with blunt ends is less efficient, needing random collisions.

Introduction of DNA into Living Cells

• Transformation is the uptake of DNA by bacterial cells under certain circumstances and conditions.
• Bacteria need to be modified, or made competent, to efficiently take up DNA.

Transformation

• Not all types of bacteria are equally as efficient to take up DNA under normal conditions.
• Bacteria require modification in the lab to enhance their DNA uptake abilities.

Selection for Transformed Cells

• It is important to distinguish cells that have taken up a plasmid from those that have not.
• This differentiation is usually done by using antibiotic resistance or by using a special enzyme screening mechanism.

Recombinant Selection with pBR322-Insertional Inactivation of an Antibiotic Resistance Gene

• Recombinant selection with pBR322 usually involves inactivation of an antibiotic resistance gene by insertion of foreign DNA.

After Transformation

• Transformants and non-transformants are easily differentiated and separated from each other.

To Identify Recombinants

• Replica plating technique is used to identify cells that do or do not grow after incubation on a certain medium.
• Cells that do not grow are the ones where the DNA of interest has been inserted into the inactivation gene of the cloning vector.

Insertional Inactivation

• Insertional inactivation is usually applied in gene cloning to distinguish between recombinants and non-recombinants.

Insertional inactivation does not always involve antibiotic resistance - Lac Selection/Blue-White Screen

• Insertional inactivation can also be used with the Lac Selection system, a method that uses a gene that codes for part of the enzyme b-galactosidase, which is used to distinguish recombinants from non-recombinants.

B-galactosidase

• The enzyme b-galactosidase breaks down X-gal into a blue product, while a non-recombinant, functional lacZ gene will lead to a blue color.
• A recombinant will not be able to produce B-galactosidase, leading to a white color.

Transfection

• Transfection is a process in which genetic material such as DNA and/or RNA is introduced into mammalian cells.

Transfection Methods

• There are different methods for transfecting mammalian cells, like liposome transfection, viral transduction, electroporation, optoporation, and microinjection.

Cloning Serves Two Main Purposes

• Cloning enables the production of numerous recombinant DNA molecules from a limited starting material.
• Cloning provides a pure sample of an individual gene, separated from other genes.

Purification

• Cloning techniques allow for the separation and purification of specific genes from a complex sample of DNA.