Recombinant DNA Technology and Cloning

Recombinant DNA Technology

• Recombinant DNA technology, also known as genetic engineering, involves techniques to identify, isolate, and recombine DNA from different sources to introduce new traits into an organism.
• Paul Berg, Herbert W. Boyer, and Stanley N. Cohen greatly contributed to the method in which genetic material from one organism is artificially integrated into another oragnism.
• The first recombinant DNA molecules combined DNA from the SV40 virus and lambda phage.
• Genetically engineered DNA molecules can be developed and cloned in foreign cells.
• Cloning refers to the generation of identical DNA molecules.

DNA Cloning

• DNA cloning is the production of numerous identical DNA molecules derived from a single ancestral DNA molecule.
• DNA fragments are selectively amplified, leading to an increase in the copy number of specific DNA sequences.
• This typically involves multiple rounds of DNA replication with a DNA polymerase.
• Two main DNA cloning approaches include Cell-based and cell-free DNA cloning.

Cell-Based DNA Cloning

• This was the earliest DNA cloning method developed.
• Desired DNA fragments attach to DNA molecules capable of independent replication.
• The resulting recombinant DNA molecules are transferred into host cells to be selectively replicated.
• Process involves:
  • Construction of recombinant DNA molecules: Autonomously replicating DNA molecules combine with desired DNA fragments, forming hybrid molecules (chimeras).
  • In vitro covalent ligation joins target DNA fragments to a cloning vector, which acts as a replicon (any DNA molecule capable of independent DNA replication). The target DNA and cloning vectors are cut with specific restriction endonucleases before being joined by DNA ligase.
  • Transformation: DNA molecules are transferred into host cells (often bacterial or yeast cells), where chosen replicon replicate independently without the host cell chromosome(s).
  • Selection of transformed cells involves identifying and isolating cells that successfully took up DNA molecules through the process of transformation.
  • Selection including recombinant DNA distinguishes and keeps transformed cells with recombinant DNA, that have taken up a recombinant DNA molecule containing the desired DNA fragment.

Cell-Free DNA Cloning

• Polymerase chain reaction (PCR) selectively amplifies the specific target nucleotide sequence.
• PCR, developed in 1983 by Kary Mullis, is enzyme-mediated and conducted entirely in vitro.
• Utilizes short primers for copied DNA sequences to replicate DNA with rapid, inexpensive and simple steps.

Enzymes for DNA Manipulation

• Common enzymes for DNA manipulation in recombinant DNA technology are DNA polymerase, nucleases, DNA ligase, and end-modification enzymes.

DNA Polymerase

• DNA polymerase enzymes synthesize new polynucleotides using an existing DNA or RNA template.
• DNA polymerase I (Kornberg enzyme) has 3' to 5' and 5' to 3' exonuclease activities and 5' to 3' polymerase activity, used in gene manipulation.
• Reverse transcriptase (RNA-directed DNA polymerase) synthesizes DNA from RNA, discovered by Howard Temin and David Baltimore, who shared the 1975 Nobel Prize.
• Taq DNA polymerase, from Thermus aquaticus, functions at 72°C and stable above 90°C is used in PCR. Possesses 5' to 3' polymerase and exonuclease activity but lacks 3' to 5' exonuclease activity.

Nucleases

• Nucleases degrade nucleic acids by breaking the phosphodiester bonds and has two different kinds, exonucleases and endonucleases.
• Ribonucleases (RNases) attack RNA.
• Deoxyribonucleases (DNases) attack DNA.
• Exonucleases remove nucleotides from the ends of nucleic acids, attacking either the 5' or 3' end.
• Endonucleases break internal phosphodiester bonds.
• Mung bean nuclease: An endonuclease specific for single-stranded DNA and RNA, purified from mung bean sprouts. Digests single-stranded nucleic acids, but leaves double-stranded regions intact.
• S1 nuclease: An endonuclease purified from Aspergillus oryzae that degrades RNA or ssDNA but does not degrade dsDNA or RNA-DNA hybrids. Cleaves a strand opposite a nick on the complementary strand.
• RNase A: An endonuclease that digests ssRNA at the 3' end of pyrimidine residues.
• RNase H: An endonuclease which digests the RNA of an RNA-DNA heteroduplex but does not digest ss or dsRNA.
• Restriction endonucleases (restriction enzymes): Bacterial enzymes that cut dsDNA into fragments at specific nucleotide sequences (recognition or restriction sites). This term refers to the fact that these enzymes restrict the entry of foreign DNA in bacteria. They are believed to have evolved to resist viral attacks.
  • The existence of restriction enzymes was first postulated by W. Arber, who theorized their presence when bacteriophage DNA entered a host bacterium and was cut into smaller pieces.
• Hamilton Smith and his co-workers isolated restriction enzymes from Haemophilus influenzae strain Rd in 1970. The enzyme, called HindII, recognizes a six base-pair dsDNA sequence.
• After discovery of HindII restriction enzyme, EcoRI was isolated and characterized from Escherichia coli strain RY13.
• Nomenclature: The name of an abbreviation of the genus and species of the organism to three letters. A letter, number, or combination of the two to indicate the strain of the relevant species. A Roman numeral indicating the order of isolation of different restriction enzymes from the same organism.

Restriction (or recognition) sites

• Rather than cutting DNA indiscriminately, a restriction enzyme cuts only double-helical segments that contain a particular nucleotide sequence of four to eight base pairs in length, known as a restrictions site.
• These are generally palindromic sequences.
• The position at which the restriction enzyme cuts is usually shown by the symbol '/'.
• Restriction enzymes make either blunt or staggered cuts. Thus, the restriction fragments may have:
  • Blunt ends (the cleavage points occur exactly on the axis of symmetry).
  • Overhanging ends (the cleavage points do not fall on the symmetry axis, so that the resulting restriction fragments possess sticky ends or cohesive ends).
  • After the staggered cuts, the resulting restriction fragments possess so-called 5' overhangs or 3' overhangs.
• Isoschizomers, neoschizomers, and isocaudomers:
  • Isoschizomers are pairs of restriction enzymes that recognize the same recognition site and also cut at the same location.
  • Neoschizomers are enzymes that recognize the same sequence, but cut it differently.
  • Isocaudomers are enzymes that recognize slightly different sequences but produce the same ends.

Restriction-Modification system

• This contains a restriction enzyme and does not cut its own DNA because the system includes restriction endonucleases that selectively recognize specific DNA sequences (recognition sites) and cleaves the DNA having these sequences.
• In the modification system includes a modification enzyme (DNA methylase) which recognizes the recognition sites and modifies the sites by adding methyl group to one or two bases.
• Modify the existing DNA so that is will be not be cleaved. Restriction endonucleases are divided into 3 types because factors.

End-Modification enzymes

• End-modification enzymes are used to make changes to the ends of nucleic acids.
• Terminal deoxynucleotidyl transferase (TdT) synthesizes a new DNA polynucleotide without base-pairing of the incoming nucleotides to an existing strand of DNA or RNA.
• This enzyme is used for the formation of a cohesive end by homopolymer tailing because terminal deoxynucleotidyl transferase catalyzes the addition of a series of nucleotides onto the 3'-OH termini of a dsDNA molecule.
• Alkaline phosphatase removes phosphate groups from the 5' ends of DNA molecules, to prevent ligation to one another. Bacterial alkaline phosphatase is more stable but less active
• T4 polynucleotide kinase adds phosphates to 5' ends, performing the reverse reaction to alkaline phosphatase used for end-labeling of DNA molecules. the kinase utilizes two types of reactions: forward reaction and exchange reaction.

Ligases

• They join DNA molecules forming phosphodiester bonds between nucleotides at the the ends of two different molecules, or at the two ends of a single molecule. DNA ligases commonly used in cloning experiments are those obtained from E. coli or from the bacteriophage T4.

Vectors

• A vector refers to the DNA molecules transport as transporting vehicle that carries target DNA into a host cell for the purpose of cloning and expression.
• Cloning vectors clone target DNA.
• Expression vectors are engineered so that desirable target DNA can be transcribed in RNA and translated into protein.
• Viral DNA or plasmid are generally used as a vector.
• Important features:
  • Ability to replicate in host cells is a specific sequence in DNA from where replication starts when target DNA is linked to vector containing origin of replication and desirable target DNA also starts replicating within the host cell.
  • Unique restriction sites for insertional cloning allows a target DNA to be conveniently inserted into the vector. This may be a multiple cloning site (also called polylinker site) which contains many unique restriction sites.
  • Restriction site(s) cleaved by specific restriction enzyme(s), and a target gene is ligated into the vector by DNA ligase.
• Genetic marker to select for host cells containing the vector.
• Marker genes belong to two broad categories: selectable markers and screenable markers. A selectable marker gene encodes a product for cell groth, and a screenable marker gene is a reporter gene.
• Low molecular weight that has the plasmid is more resistant to damage by shearing and is readily isolated from host cells, plasmids are usually present in multiple copies, less chance that the vector will have multiple sites for any restriction endonuclease.

Vectors for E. coli

• Cloning vectors based on plasmid DNA.
  • Plasmids are naturally occurring circular, extrachromosomal, autonomously replicating DNA, present in many prokaryotic and a few eukaryotic organisms.
  • Range in size from approximately 1 kb to over 300 kb.
  • Adapt natural plasmid molecules as cloning vectors, several modifications are normally made as an insertion of a multiple cloning site, polylinker site.
  • A widely used cloning vector is pBR322, which replicates in E. coli having genes conferring ampicillin resistance and tetracycline resistance on its host and has single cleavage sites.
• Bacterial artificial chromosome (BAC): They are based on the F-plasmid and are maintained within E. coli as large, single-copy plasmids, capable of accommodating inserts ranging from 50 to 300 kbp .
• Vectors incorporate the F-plasmid origin of replication and genes regulating plasmid replication and copy number.
• Cloning vectors based on viral DNA:
• The gene(s) of interest are incorporated into the genome of a virus, high efficiency increases frequency than simple transformation.
• Lambda phage, a temperate phage, infects bacteria E. coli and replicates by a lytic or lysogenic pathway.
• Deletion of non-essential DNA of allows much new DNA to be added.
• Two basic types of lambda vectors are developed: insertion vectors and replacement vectors.
• In vitro packaging requires a number of different proteins coded by the 2. genome and can prepared at a high concentration from cells infected with defective a phage strains. Two different systems are in use.
• Cosmids: These are vectors that are hybrids of lambda phages and plasmids, their DNA replicate as plasmids, or can can be be packaged. A development of cosmid vectors based of 200 bp DNA in the lambda phage genome called cos site used for DNA packaging during lytic infection.
• Fosmids (based on the bacterial F-plasmid) contain lower copy number in E. coli and can hold DNA inserts of up to 40 kb in size.
• Cloning vectors based on M13 Phage the cloning in DNA sequencing is espeically important.
• Cloning vectors based on P1 phage PAC (a P1-derived artifical chromosome) are constructs derived from P1 and has largest genome, and contains essential repication.

Cloning vectors for yeast, S. cerevisiae

• Shuttle vectors are capable of propagating between two different organisms with unique origins of replication for each cell.
  • Yeast Episomal Plasmids (YEP) for replication are based on the endogenous yeast.
  • Yeast Integrative Plasmids (YIp) integration carry a bacterial plasmid and are stable and does not contain 2micro plasmid,
  • Yeast Replicating Plasmids (YRp) capable of are capable of autonomous replication.
  • Yeast Artificial Chromosomes (YAC) : are allows cloning. They are essential pBR322 plasmid functional for protection like telomeres

Vectors for plants

• Vectors for plants are based on either plasmid or viral genome:
  • The Plasmids in species Agrobacterium used plants transform them.
• Plasmid based vector:
  • Ti plasmids and Ri Plasmids are considered natrual genetic engineers.
• Ti plasmid based vector: It is a process of plant genetic mediated to get T-DNA. The second component is the vir genes for expression.
  • carries also carries genes for differentiation and opines that are made to be used and catabolized. This allows genes for the transfer of T-DNA by the operons. This signals and binds.

Vectors for animals

• Vectors for insects:
  • P element and P DNA.
• Vectors for Mammals:
  • Genome of many virus and they create vectors. At present, retroviruses are more common.

Introduction of DNA into the Host Cells

• Introduction of DNA involves two different types:
  • Chemical transformation method involving uptake DNA that is competent and by treatment for competancy is a ohysiologic state that treat to cell, CaCl₂ is introduced (or use an electric pulse).
  • Electroporation also can be achieved throughout of electrical use of electrical pulses.
• Plant Cells include exogenous DNA includes a transformation gene by gene transfer in two different methods:
  • Vector-Mediated Methods and explaition bacteria. -Agrobacterium has transformed with a step process to get the t-dna and protein for expression.

Selectable and screenable markers

• Those two types of markers help transform.
• Negative vs Positive is what will make them grow to find the best result.
• Several genes help with results as reporter results.
• LacZ, CAT, GFP are best to visualize results with the best markers.

Selection of transformed bacterial cells

• This is must because the selection of DNA creates antibiotics and their actions can be manipulated to be resistant after transformation.
• B-galactose gene complementation is the best for selection because it can indicate a blue print depending on the type of DNA.

Expression Vector

• It is a factor that regulates expressions, and vector carries a prompter for transcription of the genes.
• Regulation helps by induction or response. Regulatory are the important components for expression.
• Expression system is important, for example in the prokaryotic system E.COLI is commonly the model, for easy production.
  • For prokaryotic expression problems can develop, for example introns in protein, processing.
  • Eukaryotic systems can be changed to control issues and give tagged protein .