DNA Cloning: Principles and Vectors

• Molecular methods include basic principles, applications, and health and safety considerations.
• Techniques covered are extraction/purification of DNA/RNA, DNA cloning, DNA sequencing, PCR & RT-PCR, and gel electrophoresis.

DNA Cloning

• DNA cloning reproduces DNA fragments through cell-based approach or polymerase chain reaction (PCR).
• A vector is required to carry the DNA fragment of interest into a host cell.
• It allows copies of a specific DNA or RNA sequence for unlimited production.
• It serves as part of genetic engineering experiments like production of DNA libraries, PCR, and DNA sequencing.
• Enables massive amplification and stable propagation of DNA sequences.
• Amplified DNA molecule can be studied, sequenced, manipulated, mutated, engineered, or expressed for protein generation.
• Cloning requires a plasmid as a vector.

Cloning Vectors

• Cloning vectors are DNA molecules used to transport cloned sequences between biological hosts and test tubes.
• Common properties include autonomous replication, a genetic marker for selection, unique restriction sites for insert DNA cloning, and a minimal amount of nonessential DNA to optimize cloning.
• Vectors require high frequency of uptake into cells, high copy number, unique cloning sites, and at least one selectable marker.
• Different types of cloning vectors serve different cloning experiments depending on size and type of DNA to be cloned.
• Plasmids, bacteriophages, and cosmids are cloning vectors.
• Plasmids are circular extra-chromosomal DNA with a high copy number (50-200/cell)
• Typically 4-8 kbp and can accommodate 4-6 kbp of DNA, with 1-2 selectable markers and unique cloning sites.
• Plasmid vectors contain a replication origin sequence (ORI) and a gene that permits selection, which encodes the enzyme, B-lactamase, that inactivates ampicillin.
• Exogenous DNA can be inserted into the bracketed region.

Selectable Markers

• Selective marker is required for maintenance of plasmid in cells and is usually a gene for antibiotic resistance.
• Examples are tetracycline (tet), ampicillin (amp'), or an enzyme that gives a color change, such as lacZ'.
• It allows selection of transformants (cells that have taken up a plasmid).
• Only cells containing plasmids with the selectable marker can survive under selective conditions.
• E. coli can take up a plasmid under certain conditions called transformation.
• If the strain of E. coli is sensitive to ampicillin and the plasmid contains the amp' gene, only successful transformants will be ampicillin-resistant.

Multiple Cloning Site (MCS)

• Multiple cloning site (MCS), aka polylinker, is a DNA segment with several unique sites for restriction endonucleases located next to each other.
• MCS restriction sites are not elsewhere in the plasmid.
• Cutting plasmids at one of the restriction sites within the MCS does not disrupt essential features of the vector.
• Recombinant bacteria manufacture insulin plasmid vector with origins of replication (ori), multiple cloning site, and ampicillin resistance gene.

pGLO Plasmid

• pGLO allows transformed bacteria to survive on plates with ampicillin with arac gene for the protein AraC controlling the GFP gene light and on/off switch.
• It also features the GFP gene for green fluorescent protein.

Plasmid Cloning Steps

1. Isolate plasmid and human DNA.
2. Cut both DNA samples with the same restriction enzyme.
3. Mix the DNAs; they join by base pairing creating recombinant and nonrecombinant plasmids.
4. Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene.
5. Recombinant bacteria manufacture insulin.

Plasmid Vectors

• Plasmid vectors clone DNA ranging in size from bp to several 1000s of bps (100bp-10kb).
• ColE1-based, pUC vehicles are commercially available ones, e.g. pGEM3, pBlueScript

DNA Cloning Process

1. Digestion of DNA sample with EcoRI enzyme
2. Digestion of plasmid DNA
3. Ligation of DNA sample and plasmid DNA with DNA ligase + ATP
4. Transformation of ligation products via heat shock or electroporation
5. Growth on agar plates

Blue vs. White Colonies

• Blue colonies represent ampicillin-resistant bacteria with vector and a functional B-galactosidase from an intact lacZ coding sequence.
• White colonies represent ampicillin-resistant bacteria with vector + insert and no lacZ alpha fragment.

PCR cloning strategies

• There are blunt-end cloning via restriction enzymes, sticky-end cloning via restriction enzymes, and TA cloning.

TA Cloning

• Resulting DNA fragments have the enzyme, Taq polymerase, which adds extra A to the end of the 3' end of blunt dsDNA
• Use a plasmid vector with T residues linked onto the 3' ends of linearised plasmid DNA.

Analyzing Cloned DNA

• Analysis determines whether the DNA is the desired piece via sequencing or restriction enzyme digestion.
• It also identifies molecular characteristics through expression or mutational analysis.

DNA Sequencing Process

• You need to have lots of DNA from PCR or cloning.
• Techniques to read the sequence are:
  • Dideoxynucleotide (“Sanger”) sequencing
  • PCR cycle sequencing
  • Next Gen sequencing

Methods of DNA Sequencing

• Several methods used are Maxam and Gilbert method and Sanger sequencing
• Sanger used everywhere and Next Gen seq used in clinical applications.
• Method depends on the production of a mixture of oligonucleotides labeled either radioactively or fluorescently.
• They have on common end & differing in length by a single nucleotide at the other end
• Oligonucleotides are separated by high resolution electrophoresis on polyacrylamide gels and the bands' position determined

The Maxam-Gilbert Technique

• Chemical degradation of Purines are used
  • Purines (A, G) damaged by dimethylsulfate
  • Results in methylation of base and heat releasing of base
  • Then alkali cleaves G and dilute acid cleaves A>G

The Original Sanger Method

• Involves random incorporation of a dideoxynucleoside triphosphates (ddNTPs) into growing DNA strand
• These are terminators
• Process requires a primer, DNA polymerase, a template, a mixture of nucleotides, and detection system, and Uses 32P-deoxynucleoside triphosphates

Chain terminators

• Includes acylovir and guanosine.

DNA cycle sequencing using dyes

• DNA cycle sequencing is automated with radioactive deoxynucleotide not used when all 4 dideoxy reactions are performed in this single tube automated approach.
• This is possible because each ddNTP is labeled with a different fluorescent dye.
• The dye corresponds to the dideoxynucleotide added to discontinue the synthesis of that fragment.
• Single tube reaction contents are loaded in a single lane for gel electrophoresis

Automated DNA sequencing

• It uses a flourimeter and computer hooked to a gel to detect/record the dyes to determine sequence per dies.

Next Gen Sequencing

• It is a third generation approach.
• Nucleotide detection occurs in a zero-mode waveguide (ZMW) as featured in PacBio sequencers.
• DNA polymerase molecules attach to the bottom of ZMW with labeled nucleotides.
• Nanopore DNA sequencing is used in ONT's MinION sequencer.
• Double-stranded DNA is denatured by a processive enzyme and passes through a biological nanopore.
• As ssDNA passes through the nanopore, bases prevent ionic flow distinctly and allowing inference of the DNA molecule via monitored current at each channel

Single-molecule real-time (SMRT) - PacBio

Nanopore - Oxford

• A protein nanopore is set in an electrically resistant polymer membrane and an ionic current is passed through via settings of voltages.
• If an analyte passes through, the events creates a characteristic disruption in the current and makes it possible to identify said molecule.

Speed and Cost of Sequencing

• Cost of sequencing the first human genome in 2003 was $2.1 billion for 13 years.
• Cost dropped to $300,000 for 3 wks in 2006, then $1000 for 10 days in 2014.
• Cost was $150 and took 19.5 hrs in 2018.