Molecular Cloning: DNA Technology and Vectors

• These study notes cover fundamental technologies in molecular cloning, including recombinant DNA technology. Molecular biotechnology involves isolating and transferring genes between organisms. The core principle is joining a DNA sequence of interest to a vector, which can be introduced into a host.

Molecular Cloning Essentials

• A DNA fragment (insert) is cloned into a vector for cell delivery.
• Procedures are needed to obtain the insert and vector, plus specific tools.
• A suitable host cell is also needed.

Plasmid Vectors Overview

• Plasmids are small DNA molecules distinct from chromosomal DNA and can replicate independently. They are often circular and double-stranded episomes.
• They contain genes for toxin production, antibiotic resistance, enzyme production, and pathogenicity.
• A vector is a DNA molecule carrying foreign genetic material into a cell.

Plasmids and Bacterial Conjugation

• Plasmids transfer between bacteria via conjugation, requiring a pilus (a protein tube encoded by the F plasmid)
• The pilus connects donor (F+) and recipient (F-) bacteria, transferring plasmid DNA.
• The recipient converts to F+, gains the pilus, and becomes a donor
• Plasmids confer selective properties and contribute to antibiotic resistance.

Engineered Plasmids

• Plasmids serve as genetic vectors due to their transferability between bacteria and ability to carry genes.
• Ori (Origin of replication) is Required for replication initiation and copy number.
• MCS (Multicloning site) For restriction endonuclease cleavage and insert introduction.
• Promoter region is Required for transcription initiation at the MCS.
• Selectable marker: Is often an antibiotic resistance gene.

Advantages of Plasmids as Genetic Tools

• Plasmids are easy to work with, small (2,000-20,000 base pairs), and can be easily produced and modified.
• Being self-replicating, they can create many copies by growing the plasmid in bacteria.
• Plasmids are stable both as purified DNA and within bacteria.
• They have a wide range of uses, including understanding gene function, investigating promoters, and protein production.

Restriction Enzymes and Palindromes

• Restriction sites in bacterial DNA contain palindromic sequences.
• A palindrome reads the same forwards and backwards (like RADAR).
• DNA is always read from 5' to 3', with palindromes having the same sequence on complementary strands.
• Restriction endonuclease EcoRV recognizes the GATATC sequence.

Restriction Endonucleases in Molecular Biology

• Restriction endonucleases are useful tools in molecular biology, and Bacteria contain enzymes that cleave double stranded DNA at palindromic sequences, known as restriction sites.
• Modified plasmid vectors contain several restriction sites.
• They allow for cloning and analysis of specific DNA fragments.
• The frequency of restriction sites depends on the length of the restriction site.

DNA Cleavage by EcoRI

• EcoRI recognizes the sequence GAATTC and cleaves both DNA strands on the same site.
• It generates single-stranded regions at the 5' end, known as sticky ends.
• The DNA ends produced will have a 3'-OH and a 5'-P.

DNA Cleavage by PstI.

• Pstl recognizes the sequence CTGCAG and cuts both strands of double-stranded DNA (dsDNA) at the same site.
• It generates DNA ends that have single-stranded regions at the 3' end of the molecule.
• These ends are referred to as sticky ends.
• The ends of the DNA will be a 3'-OH and a 5'-P.

DNA Cleavage by SmaI

• SmaI recognizes the sequence CCCGGG and cleaves both strands of double-stranded DNA (dsDNA) at the same site.
• It generates DNA ends without single-stranded regions
• These are known as blunt ends.
• The ends of the DNA will be a 3'-OH and a 5'-P.

Bacterial Genome Defense Mechanisms

• Restriction endonucleases protect bacteria from phage infections.
• Each bacterial species has a DNA sequence-specific restriction enzyme.
• Bacteria possess DNA methylases to methylate and protect its own DNA from being cleaved.
• Phage DNA is typically not methylated, making it vulnerable.

Restriction Site Frequencies

• The frequency of restriction sites depends on the length of the recognition sequence.
• AluI sites (4 base pairs) occur approximately every 256 base pairs (4^4).
• NotI sites (8 base pairs) occur approximately every 65,536 base pairs (4^8).

Obtaining insert DNA

• There are multiple ways to obtain the insert DNA for cloning, and the most common of these include restriction digestion from genomic DNA, PCR amplification and isolation from a genomic library (already in a plasmid).
• The DNA that will be inserted into the vector will need to be isolated and purified, and which is be carried out by gel electrophoresis.

Restriction Digestion Protocol

• Restriction digestion involves using restriction enzymes to cut DNA at specific sites.
• Plasmid DNA is incubated with a restriction enzyme to create DNA fragments.
• The DNA fragment (insert) is then purified through an agarose gel.

Gel Electrophoresis Principles

• Gel electrophoresis separates DNA fragments by size.
• The process involves preparing an agarose gel, applying a voltage, and running the gel.
• A 1X TAE Buffer (Tris, Acetate, EDTA) with Ethidium Bromide is used.

Gel Electrophoresis Results

• Gel electrophoresis results in bands representing different DNA sizes.
• Relaxed and supercoiled plasmids can be distinguished, along with plasmid fragments.

Cloning strategy

• Gene is put into plasmid or Genomic DNA, before undergoing Restriction digestion* or PCR with primers.
• The DNA undergoes Gel purification, and then Ligation and Transformation.
• It tested for by electrophoresis.

Target DNA Insertion into Plasmid Vectors

• Compatible restriction sites on DNA ends allow reannealing, but DNA ligase is needed for covalent linking.
• DNA ligase links the 3'-OH of one end with the 5'-P of the other to make a single DNA molecule.

Enzymes for Cloning

• Mung bean nuclease removes single-strand overhangs, resulting in blunt ends.
• DNA polymerase can fill in 5'-overhangs to create blunt ends.
• Calf alkaline phosphatase removes 5′-P to prevent ligation.
• T4 polynucleotide kinase adds a 5'-P to facilitate ligation.

pUC19 Cloning Vector

• The pUC19 is a commonly used cloning vector, with features like a lacZ gene and multiple cloning site (MCS).
• The lacZ gene is a selective marker

Cloning into pUC19 Steps

• pUC19 and target DNA are cut with BamHI.
• The cut vector is treated with alkaline phosphatase to prevent self-ligation.
• DNA ligase joins the target DNA into the vector.

Using the lacZ Gene as a Cloning Reporter

• The lacZ gene encodes β-galactosidase, which cleaves lactose.
• Cloning in the lacZ gene allows blue/white screening.

Cloning Procedure Overview

• Digest human genomic DNA and plasmids with the same restriction endonuclease.
• Introduce fragments into plasmids at the MCS within lacZ.
• Allow compatible ends of fragments to re-anneal and ligate with DNA ligase.
• Introduce Plasmids containing DNA fragments into E. coli through transformation (heat shock).
• Plate cells on plates with antibiotic/X-Gal for identification of colonies.
• Select white colonies.

Transformation Process

• Transformation allows bacterial cells to uptake DNA.
• One method involves treating mid-log phase cells with ice-cold calcium chloride to make them competent, then exposing them to high temperature
• Electroporation is also used, subjecting bacteria to a high-voltage electric field.

Cloning Selection

• Bacteria are selected through uptake of different DNA products
• Only the proper plasmid, which contains the selectable marker will be selected.
• Bacterial colonies do not have an insert if the lacZ gene is expressed
• the lacZ gene is normally expressed generating β-galactosidase, which presence of X-Gal. giving a blue color.
• with interruption of the lacZ gene in the process the bacterial colonies have a insert, with no colony generated from the β-galactosidase, resulting in the noticable absence of the blue color.
• IPTG is used to induce the LacZ gene to create colour.

Confirmation of Cloning

• Selected colonies are picked and grown on overnight cultures to clone for it
• The DNA is then confirmed through restriction digestion or DNA sequencing.