Phage Lambda Vectors: Insertional vs. Replacement

Gene Manipulation and Recombinant DNA

• Gene manipulation involves the creation and cloning of recombinant DNA.
• Recombinant DNA is artificially created, combining DNA sequences not typically found together in nature.
• Gene manipulation encompasses techniques for creating recombinant DNA and introducing it into living cells.
• Cloning propagates recombinant DNA within a host cell, producing many copies of the same sequence.
• Cloning yields homogeneous preparations of desired DNA molecules in amounts suitable for lab experiments.

Cutting DNA Molecules

• Before 1970, methods for cleaving DNA at discrete points were unavailable.
• Available fragmentation methods before 1970 were non-specific.
• Available endonucleases had little site specificity.
• Chemical methods produced very small DNA fragments.
• Mechanical shearing was a method with limited control.
• Duplex DNA molecules, rigid enough to be broken by shear forces in solution, can be sonicated with ultrasound to reduce length to about 300 nucleotide pairs.
• High-speed stirring in a blender achieves more controlled shearing at ~8 kb mean DNA size by stirring at 1500 rpm for 30 min, with random breakage and single-stranded termini.
• Restriction endonucleases from E. coli K12 cut unmodified DNA into large, discrete fragments, recognizing a target sequence but cleaving "randomly" several kilobases away.
• An enzyme discovered in H. influenzae in 1970 recognizes a target sequence in duplex DNA, breaking the polynucleotide chain within that sequence to give discrete, defined fragments.
• Four types of restriction and modification (R-M) systems have been recognized but only Type II is widely used in gene manipulation.

Type II Endonucleases

• Type II endonucleases recognize the same target sequence, which is symmetrical.
• Type II endonucleases cleave or modify the recognition sequence.

Naming Restriction Endonucleases

• The species name of the host organism provides information about the source of restriction endonucleases.
• The first letter of the genus name and the first two letters of the specific epithet create a three-letter abbreviation.
• The particular strain identifies the organism.
• Roman numerals identify multiple R-M systems in a single host strain.
• Restriction enzymes cut DNA at rotational symmetry sites, with different enzymes recognizing different sequences.

Joining DNA Molecules

• Joining DNA fragments creates artificially recombinant molecules.
• DNA ligase covalently joins annealed cohesive ends produced by certain restriction enzymes.
• T4 phage DNA ligase catalyzes phosphodiester bond formation between blunt-ended fragments.
• Terminal deoxynucleotidyl transferase synthesizes homopolymeric 3' single-stranded tails at fragment ends.
• E. coli and phage T4 encode DNA ligase, sealing single-stranded nicks, differing in cofactor requirements where T4 enzyme requires ATP and E. coli enzyme requires NAD+.
• The cofactor forms an enzyme-AMP complex and binds to the nick (5' phosphate and 3' OH group), creating a covalent phosphodiester bond.
• This reaction, performed in vitro with purified DNA ligase, is fundamental to gene manipulation.
• The optimum temperature for nicked DNA ligation is 37°C, the hydrogen bonds between sticky ends are unstable.
• The optimum temperature for ligating cohesive termini is a compromise between enzyme action and termini association, found experimentally to be in the range 4-15°C.

Adaptors and Linkers

• Adaptors and linkers are short, double-stranded DNA molecules that permit different cleavage site interconnection.
• Linker molecules are synthetic, self-complementary decameric oligonucleotides containing sites for one or more restriction endonucleases.
• Restriction endonucleases act on linker molecules producing a sticky-ended fragment.
• If the restriction enzyme also cuts the foreign DNA at internal sites, the foreign DNA will be cloned as two or more subfragments.
• Solutions include choosing another restriction enzyme or methylating internal restriction sites with the appropriate modification methylase.
• Adaptors are chemically synthesized molecules with a preformed cohesive end, featuring a blunt end bearing a 5' phosphate group and a cohesive end that is not phosphorylated.
• The former contains blunt ends and the latter has cohesive ends.

Cloning Vectors

• An ideal cloning has the following properties;
• Origin of replication, allow the vector to replicate independently ensuring the vector (and inserted DNA fragment) is multiplied in the host cell.
• Low molecular weight, preferred because it allows for easier manipulation and more efficient insertion into the host cell. They tend to be more stable, and less prone to breakage during cloning. Selectable phenotypic traits on cells, a gene carried by the vector that confers. a selectable advantage to the host cell, allowing cells containing the vector to be indentified and isolated.
• Unique restriction sites (multiple cloning site) (MCS), allows for insertion of DNA fragement of interest, using a variety of restriction enzymes. Ensuring flexibility during the experiment. Uniqueness of the site ensures the vector is cut only at the desired location.
• Low molecular vector advantages
  • Easier to handle (resistant to damage by shearing during isolation from cells)
  • Plasmids are usually present as multple copies, allows facilitates isolation and leads to gene dosage.
  • Less changes of multiple sites for any restriction endonucleases.
  • Pieces of foreign DNA are inserted for resulting chimaeric molecules.
  • transformed into suitable recipient.

Plasmids

• Widely used as cloning vehicles (replicons, stably inherited in an extrachromosomal state).
• Exist as double-stranded circular DNA molecules. Covalently closed circles (CCC DNA): both DNA strands are intact circles in supercoiled configuration.
• Open circles (OC DNA): one strand is intact.
• Supercoiled and OC DNA separate on electrophoresis in agarose gels due to structural configurations.
• Plasmids range iin size from <1 × 10^6 to > 200 × 10^6 Da.
• Plasmids are generally dispensable; plasmids to which phenotypic traits have yet to be ascribed are cryptic plasmids.
• Can be categorized by maintenance as multiple copies per cell (relaxed plasmids) or as a limited number per cell (stringent plasmids).
• Only a small region surrounding the ori site is required for replication.
• Plasmids are deleted for replication, and foreign sequences may be added.

Host and Plasmid Range

• Determined by its ori region: Plasmids whose ori region is derived from plasmid Col E1 have a restricted host range.
• Can replicate in enteric bactiera such as E.coli and Samonella. _ -Some promiscious plasmids have a broad host range. -Plasmids will have broad host range, that encode most if not all of the protiens required for replication.
  • To express the genes the promotor must have evovled.

Plasmid Numbers

• Can vary, between plasmids by regulatory mechanism.

• Copy number, is controlled by antisense RNA.

• A prime for DNA replication is 555 base RNA molecule called RNA II, which forms as RNA- DNA hybrid.

• RNA II act as a primer by RNase H to leave a free 3' hydroxyl group.

• Replication Control, is mediated by anotehr amll (108-base) RNA molecule called RNA I, Encoded by the same origin region.

• It can interferes with the rpocessing by of RNA II by RNAse H and so replication does not ensue- since RNA I is ecoded by plasmid. . " " Plasmids must also be able to express these genes; their promoters and ribosome binding sites must have evolved so they are recognised by a variety of bacterial families. Furthermore, replication must depend almost exclusively on host-encoded factors. This will allow the plasmid to replicate in an appropriate manner in the different hosts; only the minimum functions determined by the ori region are essential. Finally, there must be no mechanism for transfer of the plasmid from one bacterial cell to another. The only way that RP4 and related plasmids can be confined to a controlled situation is to delete the transfer genes that mediate conjugation. This may also involve introduction of mutations in the genes for replication and host-range. " A plasmid-encode protein Rop a dimer.

It helps mmaintain the cpoy number - Enhances the pairing between RNA I and RNA II => Deletion of the ROP.

• The deletion, or mutatiaons result in increased copy numbers." "

• Plasmid incompatiability is the inability of two different plasmids to coexist in the same cell. " "" - These plasmids that have similar mechanincs of replication.

• pBR322 - Early cloning vector in a wildly used purpose.

• Early cloning vector used are small and natural such Col EI and PS101

• Has Genetic markers selecting transformants.

• pBR322 - best and widely used early purpose built vector.

• Contains Rp and Tc^r Genes.

  • 4326 bp
  • Compeltely seaunces

• There are >40 Enzymes to the the cleave sites.

• Has a Tcr gene, the target of its expression lies within 2 others which have promoters, and 2 genes within Clal && HinDII

• Unqiue to six enzyme with in the AP^r gene, - Cloning with pBR322, results the use of one of 1q enzymes.

• Insertaional inactivity of of the Ap ^r or the Tc^r markesrs. The DH5a strain of E. coli lacks B-galactosidase expression is also ampicillin sensititve.

• These ligation products must be screened.

• Directional cloning, is important due to the two differnet restriction sites.

"Bacteriaphage Lambada", complex virus- which is the study vector for this. "" - developes vector

"L DNa", its a linear complex ~48.5 kbp (phase Particle). At the head there are short single-stranded 5' -The DN adopts circular structure and this helps form - its, that the end contains coehise termini.

Genese, left the coventional map code is for lead and tail proteins of phage particles.

Genes of central region are in regards to reconmbination, and lyeogenation.

Much of the cetnral region. includes genes, not nessicary in order phaage to grow, and is crucial in the construction of vector.

• There are two basic ptypes - Insertiona, Replacement.
• Wildtype Lamdad, contains target sites for most commonly endonucleases.
• phages produced single target sire , insertion for insertion vectors or apir of site removing for a and Replacement.
  • it can acoomadate at onl 5% More thn its normnal DNA . The vectos are consyructed with deletions to increase the pace. 25% deleted, cannot be put in phages. An Advantage f the Stuffer fraegmt is removed, and the delted vector can give rise phages if inserted into it.

Cloning

• Directional cloning uses two different restriction sites and is important for directionality. DNA size range 36. kb - 5kb can only be packaged.

• Many, vectors for this are used used to help ECORI BAMH1.

  • Its is extended to endonucleasues the linker olecules.

Phage-vectors derivatives have been developd for several.

• to incrseas capacity of dna fragmens
• to decide to select recombinant -t o allow to prepare and copy traacription.
• t o develop vectosr for insertaion if enukatoyic dna, that such expression fo the dna* the E.coli fusion proy peptide - Such an expression vector antibody is screening.

The maximum to be attatined with the vector is type.

• and the incentive for the removal. of the stuffer. Genetic Selection is made Sl- Phenotype. E coli strain, cannot be gentic aphsge. "

"" So, vecors, include with teh region, the recombinaants(with stufrers-repalced forgin DNa) ar ephepypcail Spiv-e

-Deletioon of Game gene consequences . The gam produces neccisary DNA replication to roll. canot be used for packaging in pahge heads.

• However, game phage do fornm the the paques in the circle to
• E.coli* and *red" recombination and acircual DNA Molecules t "" - phgae forms miltiumers.

"recent genertation vextorys in in "" to "" "phages" with inserts can be selected on the basis .

Insertional vectors integrate directly, while replacement vectors swap a stuffer fragment for foreign DNA. Deleting lambda DNA makes room for inserts. Stuffer removal in replacement vectors allows larger DNA fragments to be cloned.