Topic 4: Basic Principles of Recombinant DNA Technology

DNA Cloning

The process of producing multiple copies of specific DNA fragments, typically by inserting them into bacteria for replication.

Restriction Endonucleases (RE)

Bacterial enzymes that act as "molecular scissors," cutting double-stranded DNA at specific recognition sequences usually 4–8 base pairs long.

DNA Ligase

The "molecular glue" that joins DNA fragments together by reforming covalent phosphodiester bonds; the process requires ATP and Magnesium (Mg2+).

Plasmids

Small, circular, extrachromosomal double-stranded DNA molecules found in bacteria that are often modified to serve as vectors for carrying foreign genes.

DNA Methylases

Enzymes that add methyl groups (−CH3​) to a bacterium's own DNA to protect it from being digested by its own restriction enzymes.

Isoschizomers vs. Neoschizomers

Isoschizomers are different enzymes that cleave the exact same site (e.g., SphI and BbuI); Neoschizomers recognize the same site but cleave it at different positions.

Star Activity

A loss of enzyme specificity under non-optimal conditions (like high pH or low ionic strength) where the enzyme cuts at sequences similar to, but not exactly, its cognate site.

Sticky (Cohesive) Ends

Overhanging DNA ends created by some restriction enzymes; they are preferred for cloning because they join more quickly and create stronger bonds than blunt ends.

Palindromic Sequences

The type of DNA sequence recognised by restriction enzymes, which reads the same in the 5’ to 3’ direction on both complementary strands (e.g., 5’ GAATTC 3’).

Transformation

The biological process where a host cell, such as E. coli, takes up foreign DNA (like a recombinant plasmid) from its environment.

Competent Cells

Bacterial cells that have been treated with chemicals (like Calcium Chloride) to make their membranes porous and capable of taking up DNA.

Selectable Markers

Genes carried on a plasmid (like antibiotic resistance) that allow researchers to identify and select only the bacteria that successfully took up the plasmid.

Multiple Cloning Site (MCS)

A deliberate region within a vector containing unique recognition sites for many different restriction enzymes, providing flexibility for gene insertion.

Blue-White Selection (lacZ)

A screening method where foreign DNA is inserted into the lacZ gene; successful recombinant colonies appear white (gene disrupted), while non-recombinant colonies appear blue.

Genomic Library

A collection of clones containing DNA fragments that represent the entire genome of an organism, including both coding (exons) and non-coding (introns) regions.

cDNA Library

A library made from mRNA using reverse transcriptase; it contains only the coding sequences (exons) of genes that were actively being expressed by the source cell.

Reverse Transcriptase

An enzyme isolated from retroviruses used to synthesize a complementary DNA (cDNA) strand from an RNA template.

Expression Library

A specialised library where cDNA is inserted into vectors containing a bacterial promoter, allowing the host cell actually to produce the protein encoded by the gene.

DNA Probe

A short, single-stranded, labelled piece of DNA complementary to a target sequence; it is used in hybridisation to identify specific clones in a library. hybridisation

Antibody Screening

A method used with expression libraries to identify a specific clone by using labelled antibodies that bind to the target protein produced by the bacteria.

Plaque

A clear area on a bacterial lawn where bacteriophages (viral vectors) have infected and killed the host cells; used for screening larger genomic inserts.

Tools of Recombinant DNA Technology

Restriction endonucleases (enzymes)- enzymes from bacteria
• Agarose gel electrophoresis
• Plasmids (small circular dsDNA)
• DNA ligase
• Escherichia coli (E. coli)

What are the four stages of inserting a DNA fragment into a plasmid?

1. Cutting: Both the source DNA (containing the gene of interest) and the plasmid vector are digested using the same restriction enzyme to ensure they have compatible, complementary ends.

2. Ligation: The DNA fragments are mixed together with DNA ligase, which acts as "molecular glue" to reform phosphodiester bonds and join the pieces into a single circular recombinant molecule.

3. Transformation: The recombinant DNA is transferred into a host cell, typically E. coli. This often involves making the bacteria "competent" (porous) using chemicals like Calcium Chloride and a brief heat shock.

4. Selection: Identifying the host cells that successfully took up the recombinant plasmid. This is done using selectable markers (such as antibiotic resistance) and screening methods like blue-white selection to distinguish recombinant colonies from non-recombinant ones.

How is antibiotic selection used to identify E. coli that have successfully taken up a plasmid?

• Addition: Recombinant plasmids (carrying an antibiotic resistance gene like ampR) are added to a population of E. coli.

• Plating: The bacteria are plated onto agar medium containing the corresponding antibiotic, such as ampicillin.

• Killed: E. coli that did not take up a plasmid lack resistance and are killed by the ampicillin in the agar.

• Survival: E. coli that successfully took up a viable plasmid survive and form colonies because the plasmid provides the necessary antibiotic resistance.

Bacterial Plasmid Vectors

Circular DNA vectors with a maximum insert size of ~6–12 kb. Used for DNA cloning and protein expression, but limited by small insert size and replication restricted to bacteria.

Bacteriophage Vectors (Lambda)

Linear vectors with a maximum insert size of ~25 kb. They are commonly used for cDNA and genomic libraries; for Lambda, non-essential DNA is removed to make room for the 25 kb insert.

Cosmid

Circular vectors with a maximum insert size of ~35 kb. They are used for cloning large DNA fragments but are limited by phage packaging restrictions and cannot be replicated in mammalian cells.

Bacterial Artificial Chromosome (BAC)

Circular vectors capable of carrying large inserts of approximately 300 kb. They are ideal for genomic libraries but cannot be used for protein expression and are restricted to bacterial hosts.

Yeast Artificial Chromosome (YAC)

Vectors with the largest capacity, taking inserts from 200 to 2,000 kb. They are used for very large genomic fragments but must be grown in yeast and cannot be used in bacteria.

Ti Vectors

Circular vectors used specifically for gene transfer in plants. Their insert size varies, and they are limited by a random distribution of restriction sites and difficulty in manipulation due to their large size.

Antibody Screening

This is used specifically for detecting proteins produced in an expression library. You use a labelled antibody (e.g., with a green fluorescent tag) that binds only to its specific target protein.

Hybridisation Screening

This is used for detecting DNA sequences in a genomic library or a non-expression cDNA library. Instead of an antibody, you use a labeled DNA probe—a short, single-stranded piece of DNA complementary to your target gene.