Information

What can cause incompatible sticky ends to be ligated?


Actual question

I have reason to believe (details see below) that in a ligation I carried out, an EcoRI sticky end (EcoRI: G'AATT_C) and an XmaI sticky end (XmaI: C'CCGG_G) were somehow ligated together, a process during which at least the EcoRI site was lost: the plasmid had a BamHI site very near the XmaI site, and a BamHI/EcoRI digest produced only one band compared to three bands in the undigested control (three conformations of circular plasmid).

How could ends as incompatible as EcoRI and XmaI be ligated?

Detailed Background or: Why I believe incompatible sites were ligated

This is chronologically further to: What are common causes of unexpected ligation products?. Briefly: I digested two plasmids, one with EcoRI and XmaI (p1), the other with EcoRI and AgeI (p2) [XmaI and AgeI produce compatible sticky ends], then carried out a ligation between a 1.4kb insert isolated from p2 (structure EcoRI-1.4kb-AgeI) into the 3.4kb backbone isolated from p1 (structure XmaI-20bp-BamHI-3.4kb-EcoRI). After transformation, I tested the ligation product by digest with EcoRI and BamHI, which should produce again 3.4kb + 1.4kb. The gel was poor quality but allowed the conclusion that there were different ligation products.

Repeating the EcoRI/BamHI digest and gel more carefully showed there were only two variants: Variant A (8 colonies) produced the two expected bands 3.4kb and 1.4kb. Variant B (4 colonies) only produced a clear 3.4kb band, nothing else visible in the lane: in other words, it only carried either an EcoRI or a BamHI site and was in total smaller than variant A (confirmed by undigested controls). Thus, I conclude that B was simply the same 3.4kb backbone, re-ligated without the 1.4kb insert. Since the BamHI site was untouched in the backbone during the preparatory EcoRI/XmaI digestion, I assume that the EcoRI site was not recovered during the ligation of variant B.

(The ligation was carried out overnight at 16 deg C, using Promega T4 DNA ligase and buffer and a molar ratio of 3:1 insert:vector at a total volume of 20uL. The E.coli for transformation were Invitrogen OneShot Stbl3 and have been routinely and successfully used in the lab for a long time. The analytical digestion used Promega EcoRI and BamHI in Buffer Multi-Core, which Promega claims offers 75-100% efficiency for both enzymes. Incubation was 1.5h at 37°C.)


The answer from @Armatus got me looking at star activities again. EcoRI is prone to exhibit star activity in non-optimal buffers. In the case of this enzyme this tends to be a relaxation of site specificity, so HAATTC and GAATTD would be possible cut sites. The cleavage at such sites produces the standard sticky end.

So, what if there is a star site near to the XmaI site such that a digest at the true EcoRI site and also at the star site removes the XmaI site and so allows recircularisation. If the configuration was, for example:

… GAATTC… [XmnI].GAATTD… GGATCC…

the ligated product would have neither EcoRI nor XmnI sites.


My best guess to explain this phenomenon has been spontaneous degradation of the sticky ends, followed by blunt-ended ligation.

Alternatively, single-stranded exonuclease activity either from a contaminating enzyme or from EcoRI/XmaI/AgeI could be imaginable. For example, using buffer Multi-Core is discouraged for EcoRI due to potential STAR activity - maybe it developed exonuclease activity and some plasmids ended up blunt-ended before the ligation.


I want to add an extra explanation. By personal experience I can tell that incompatible sticky-ends can ligate in some conditions. The efficiency will be lower than using compatible ends but still, some molecule will ligate if the single-stranded ends can anneal just enough for the ligase to join the DNA backbone. This can especially happen at lower temperatures where the miss-annealing of the sticky ends is more stable than at higher temperatures.

The OP states:

The ligation was carried out overnight at 16 deg C.

This condition is enough to allow incompatible sticky ends to anneal often enough to get ligated. I propose the OP to run a comparative experiment to check the efficiency of incompatible-ends ligation by running one ligation at 37C for 15min and one at 16C overnight. I expect almost no background ligation to occur in the first case.


XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

Present address: Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, 1124 W Carson Street, Torrance, CA 90502, USA

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

Present address: Division of Medical Genetics, Department of Pediatrics, Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, 1124 W Carson Street, Torrance, CA 90502, USA

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA

Department of Biological Sciences, Los Angeles, CA, USA

  • Jiafeng Gu 1, 2 ,
  • Haihui Lu 1 ,
  • Brigette Tippin 2 ,
  • Noriko Shimazaki 1 ,
  • Myron F Goodman 2 and
  • Michael R Lieber 1, 2
  • 1 Departments of Pathology, Biochemistry and Molecular Biology, Molecular Microbiology and Immunology, and Biological Sciences, USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, USA
  • 2 Department of Biological Sciences, Los Angeles, CA, USA

*Corresponding author. Norris Cancer Ctr., Rm. 5428, University of Southern California, 1441 Eastlake Ave., MC 9176, Los Angeles, CA 90033, USA. Tel.: +1 323 865 0568 Fax: +1 323 865 3019 E-mail: [email protected]

XRCC4:DNA ligase IV can ligate incompatible DNA ends and can ligate across gaps

XRCC4 and DNA ligase IV form a complex that is essential for the repair of all double-strand DNA breaks by the nonhomologous DNA end joining pathway in eukaryotes. We find here that human XRCC4:DNA ligase IV can ligate two double-strand DNA ends that have fully incompatible short 3′ overhang configurations with no potential for base pairing. Moreover, at DNA ends that share 1–4 annealed base pairs, XRCC4:DNA ligase IV can ligate across gaps of 1 nt. Ku can stimulate the joining, but is not essential when there is some terminal annealing. Polymerase mu can add nucleotides in a template-independent manner under physiological conditions and the subset of ends that thereby gain some terminal microhomology can then be ligated. Hence, annealing at sites of microhomology is very important, but the flexibility of the ligase complex is paramount in nonhomologous DNA end joining. These observations provide an explanation for several in vivo observations that were difficult to understand previously.


Genetic Engineering: History, Molecular Tools, and Everything Else

Genetic engineering primarily involves the manipulation of genetic material (DNA) to achieve the desired goal in a pre-determined way.

Some other terms are also in common use to describe genetic engineering. i. Gene manipulation ii. Recombinant DNA (rDNA) technology iii. Gene cloning (molecular cloning) iv. Genetic modifications v. New genetics.

Brief History of Recombinant DNA Technology:

The present day DNA technology has its roots in the experiments performed by Boyer and Cohen in 1973. In their experiments, they successfully recombined two plasmids (pSC 101 and pSC 102) and cloned the new plasmid in E.coli. Plasmid pSC 101 possesses a gene resistant to antibiotic tetracycline while plasmid pSC 102 contains a gene resistant to another antibiotic kanamycin. The newly developed recombined plasmid when incorporated into the bacteria exhibited resistance to both the antibiotics-tetracycline and kanamycin.

The second set of experiments of Boyer and Cohen were more organized. A gene encoding a protein (required to form rRNA) was isolated from the cells of African clawed frog Xenophs laevis, by use of a restriction endonuclease enzyme (ECoRI). The same enzyme was used to cut open plasmid pSC 101 DNA. Frog DNA fragments and plasmid DNA fragments were mixed, and pairing occurred between the complementary base pairs.

By the addition of the enzyme DNA ligase, a recombined plasmid DNA was developed. These new plasmids, when introduced into E.coli, and grown on a nutrient medium resulted in the production of an extra protein (i.e. the frog protein). Thus, the genes of a frog could be successfully transplanted, and expressed in E.coli. This made the real beginning of modern rDNA technology and laid foundations for the present day molecular biotechnology.

Some biotechnologists who admire Boyer- Cohen experiments divide the subject into two chronological categories:

1. BBC-biotechnology Before Boyer and Cohen.

2. ABC-biotechnology After Boyer and Cohen.

More information on the historical developments of genetic engineering and biotechnology is given under the scope of biotechnology.

An outline of recombinant DNA technology:

There are many diverse and complex techniques involved in gene manipulation. However, the basic principles of recombinant DNA technology are reasonably simple, and broadly involve the following stages (Fig. 6.1).

1. Generation of DNA fragments and selection of the desired piece of DNA (e.g. a human gene).

2. Insertion of the selected DNA into a cloning vector (e.g. a plasmid) to create a recombinant DNA or chimeric DNA (Chimera is a monster in Greek mythology that has a lion’s head, a goat’s body and a serpent’s tail. This may be comparable to Narasimha in Indian mythology).

3. Introduction of the recombinant vectors into host cells (e.g. bacteria).

4. Multiplication and selection of clones containing the recombinant molecules.

5. Expression of the gene to produce the desired product.

Recombinant DNA technology with special reference to the following aspects is described below:

1. Molecular tools of genetic engineering.

2. Host cells-the factories of cloning.

3. Vectors-the cloning vehicles.

4. Methods of gene transfer.

5. Gene cloning strategies.

6. Genetic engineering guidelines.

7. The future of genetic engineering.

Molecular Tools of Genetic Engineering:

An engineer is a person who designs, constructs (e.g. bridges, canals, and railways) and manipulates according to a set plan. The term genetic engineer may be appropriate for an individual who is involved in genetic manipulations. The genetic engineer’s toolkit or molecular tools namely the enzymes most commonly used in recombinant DNA experiments are briefly described.

Restriction Endonucleases— DNA Cutting Enzymes:

Restriction endonucleases are one of the most important groups of enzymes for the manipulation of DNA. These are the bacterial enzymes that can cut/split DNA (from any source) at specific sites. They were first discovered in E.coli restricting the replication of bacteriophages, by cutting the viral DNA (The host E.coli DNA is protected from cleavage by addition of methyl groups). Thus, the enzymes that restrict the viral replication are known as restriction enzymes or restriction endonucleases.

Hundreds of restriction endonucleases have been isolated from bacteria, and some of them are commercially available. The progress and growth of biotechnology is unimaginable without the availability of restriction enzymes.

Restriction endonucleases are named by a standard procedure, with particular reference to the bacteria from which they are isolated. The first letter (in italics) of the enzymes indicates the genus name, followed by the first two letters (also in italics) of the species, then comes the strain of the organism and finally a Roman numeral indicating the order of discovery. A couple of examples are given below.

EcoRI is from Escherichia (E) coli (co), strain Ry13 (R) and first endonuclease (I) to be discovered. Hindlll is from Haemophilus (H) influenza (in), strain Rd (d) and the third endonucleases (III) to be discovered.

Types of endonucleases:

At least 4 different types of restriction endonucleases are known-type 1 (e.g Ecok12), type II (e.g. EcoRI), type III (e.g. EcoPI) and type IIs. Their characteristic features are given in Table 6.1. Among these, type II restriction endonucleases are most commonly used in gene cloning.

Recognition sequences:

Recognition sequence is the site where the DNA is cut by a restriction endonuclease. Restriction endonucleases can specifically recognize DNA with a particular sequence of 4-8 nucleotides and cleave. Each recognition sequence has two fold rotational symmetry i.e. the same nucleotide sequence occurs on both strands of DNA which run in opposite direction (Table 6.2). Such sequences are referred to as palindromes, since they read similar in both directions (forwards and backwards).

Cleavage patterns:

Majority of restriction endonucleases (particularly type II) cut DNA at defined sites within recognition sequence. A selected list of enzymes, recognition sequences, and their products formed is given in Table 6.2. The cut DNA fragments by restriction endonucleases may have mostly sticky ends (cohesive ends) or blunt ends, as given in Table 6.2. DNA fragments with sticky ends are particularly useful for recombinant DNA experiments. This is because the single-stranded sticky DNA ends can easily pair with any other DNA fragment having complementary sticky ends.

DNA Ligases —DNA Joining Enzymes:

The cut DNA fragments are covalently joined together by DNA ligases. These enzymes were originally isolated from viruses. They also occur in E.coli and eukaryotic cells. DNA ligases actively participated in cellular DNA repair process.

The action of DNA ligases is absolutely required to permanently hold DNA pieces. This is so since the hydrogen bonds formed between the complementary bases (of DNA strands) are not strong enough to hold the strands together. DNA ligase joins (seals) the DNA fragments by forming a phosphodiester bond between the phosphate group of 5′-carbon of one deoxyribose with the hydroxyl group 3′-carbon of another deoxyribose (Fig. 6.2).

Phage T4 DNA ligase requires ATP as a cofactor while E.coli DNA ligase is dependent on NAD + . In each case, the cofactor (ATP or NAD + ) is split to form an enzyme—AMP complex that brings about the formation of phosphodiester bond. The action of DNA ligase is the ultimate step in the formation of a recombinant DNA molecule.

Homo-polymer tailing:

The complementary DNA strands can be joined together by annealing. This principle is utilized in homo-polymer tailing. The technique involves the addition of oligo (dA) to 3′-ends of some DNA molecules and the addition of oligo (dT) to 3′-ends of other molecules. The homo-polymer extensions (by adding 10-40 residues) can be synthesized by using terminal deoxynucleotidyltransferase (of calf thymus). Homo-polymer tailing, achieved by annealing is illustrated in Fig. 6.3.

Linkers and adaptors:

Linkers and adaptors are chemically synthesized, short, double-stranded DNA molecules. Linkers possess restriction enzyme cleavage sites. They can be ligated to blunt ends of any DNA molecule and cut with specific restriction enzymes to produce DNA fragments with sticky ends (Fig. 6.4). Adaptors contain preformed sticky or cohesive ends. They are useful to be ligated to DNA fragments with blunt ends. The DNA fragments held to linkers or adaptors are finally ligated to vector DNA molecules (Fig 6.4).

Alkaline Phosphatase:

Alkaline phosphatase is an enzyme involved in the removal of phosphate groups. This enzyme is useful to prevent the unwanted ligation of DNA molecules which is a frequent problem encountered in cloning experiments. When the linear vector plasmid DNA is treated with alkaline phosphatase, the 5′-terminal phosphate is removed (Fig 6.5). This prevents both re-circularization and plasmid DNA dimer formation. It is now possible to insert the foreign DNA through the participation of DNA ligase.

DNA Modifying Enzymes:

Some authors prefer to use the broad term DNA modifying enzymes to all the enzymes involved in recombinant DNA technology. These enzymes represent the cutting and joining functions in DNA manipulation. They are broadly categorized as nucleases, polymerases and enzymes modifying ends of DNA molecules, and briefly described below, and illustrated in Fig. 6.6.

Nucleases are the enzymes that break the phosphodiester bonds (that hold nucleotides together) of DNA. Endonucleases act on the internal phosphodiester bonds while exonucleases degrade DNA from the terminal ends (Fig 6.6A). Restriction endonucleases, described already, are good examples of endonucleases. Some other examples of endo- and exonucleases are listed.

i. Nuclease S1 specifically acts on single-stranded DNA or RNA molecules.

ii. Deoxyribonuclease I (DNase I) cuts either single or double-stranded DNA molecules at random sites.

i. Exonuclease III cuts DNA and generates molecules with protruding 5′-ends.

ii. Nuclease Bal 31 is a fast acting 3′-exonuclease. Its action is usually coupled with slow acting endonucleases.

A diagrammatic representation of the action of endo-and exonucleases is given in Fig. 6.7.

Besides the DNA cutting enzymes, there are RNA specific nucleases, which are referred to as ribonucleases (RNases).

The groups of enzymes that catalyse the synthesis of nucleic acid molecules are collectively referred to as polymerases. It is customary to use the name of the nucleic acid template on which the polymerase acts (Fig. 6.6B). The three important polymerases are given below.

a. DNA-dependent DNA polymerase that copies DNA from DNA.

b. RNA-dependent DNA polymerase (reverse transcriptase) that synthesizes DNA from RNA.

c. DNA-dependent RNA polymerase that produces RNA from DNA.

Enzymes modifying the ends of DNA:

There are certain enzymes that act on the terminal ends of DNA and modify these molecules. The important ones are listed.

a. Alkaline phosphatase that removes the terminal phosphate group (see Fig. 6.5).

b. Polynucleotide kinase involved in the addition of phosphate groups.

c. Terminal transferase (also called terminal deoxynucleotidyl transferase) repeatedly adds nucleotides to any available 3′-terminal ends, the most suitable being the protruding 3′-ends. This enzyme is particularly useful to add homo-polymer tails prior to the construction of recombinant DNA molecules.

d. The most commonly used enzymes in recombinant DNA technology/genetic engineering are listed in Table 6.3.

Host Cells —The Factories of Cloning:

The hosts are the living systems or cells in which the carrier of recombinant DNA molecule or vector can be propagated. There are different types of host cells-prokaryotic (bacteria) and eukaryotic (fungi, animals and plants). Some examples of host cells used in genetic engineering are given in Table 6.4.

Host cells, besides effectively incorporating the vector’s genetic material, must be conveniently cultivated in the laboratory to collect the products. In general, microorganisms are preferred as host cells, since they multiply faster compared to cells of higher organism (plants or animals).

Prokaryotic Hosts:

Escherichia coli:

The bacterium, Escherichia coli was the first organism used in the DNA technology experiments and continues to be the host of choice by many workers. Undoubtedly, E.coli, the simplest Gram negative bacterium (a common bacterium of human and animal intestine), has played a key role in the development of present day biotechnology.

Under suitable environment, E.coli can double in number every 20 minutes. Thus, as the bacteria multiply, their plasmids (along with foreign DNA) also multiply to produce millions of copies, referred to as colony or in short clone. The term clone is broadly used to a mass of cells, organisms or genes that are produced by multiplication of a single cell, organism or gene.

Limitations of E. coli:

There are certain limitations in using E.coli as a host. These include- causation of diarrhea by some strains, formation of endotoxins that are toxic, and a low export ability of proteins from the cell. Another major drawback is that E.coli (or even other prokaryotic organisms) cannot perform post-translational modifications.

Bacillus subtilis:

Bacillus subtilis is a rod shaped non-pathogenic bacterium. It has been used as a host in industry for the production of enzymes, antibiotics, insecticides etc. Some workers consider B.subtilis as an alternative to E.coli.

Eukaryotic Hosts:

Eukaryotic organisms are preferred to produce human proteins since these hosts with complex structure (with distinct organelles) are more suitable to synthesize complex proteins. The most commonly used eukaryotic organism is the yeast, Saccharomyces cerevisiae. It is a non-pathogenic organism routinely used in brewing and baking industry. Certain fungi have also been used in gene cloning experiments.

Mammalian cells:

Despite the practical difficulties to work with and high cost factor, mammalian cells (such as mouse cells) are also employed as hosts. The advantage is that certain complex proteins which cannot be synthesized by bacteria can be produced by mammalian cells e.g. tissue plasminogen activator. This is mainly because the mammalian cells possess the machinery to modify the protein to its final form (post-translational modifications).

It may be noted here that the gene manipulation experiments in higher animals and plants are usually carried out to alter the genetic makeup of the organism to create transgenic animals and transgenic plants rather than to isolate genes for producing specific proteins.

Vectors —The Cloning Vehicles:

Vectors are the DNA molecules, which can carry a foreign DNA fragment to be cloned. They are self- replicating in an appropriate host cell. The most important vectors are plasmids, bacteriophages, cosmids and phasmids.

Characteristics of an ideal vector:

An ideal vector should be small in size, with a single restriction endonuclease site, an origin of replication and 1-2 genetic markers (to identify recipient cells carrying vectors). Naturally occurring plasmids rarely possess all these characteristics.

Plasmids:

Plasmids are extra chromosomal, double- stranded, circular, self-replicating DNA molecules. Almost all the bacteria have plasmids containing a low copy number (1-4 per cell) or a high copy number (10-100 per cell). The size of the plasmids varies from 1 to 500 kb. Usually, plasmids contribute to about 0.5 to 5.0% of the total DNA of bacteria (Note: A few bacteria contain linear plasmids e.g. Streptomyces sp, Borella burgdorferi).

Types of plasmids:

There are many ways of grouping plasmids. They are categorized as conjugative if they carry a set of transfer genes (tra genes) that facilitates bacterial conjugation, and non-conjugative, if they do not possess such genes. Another classification is based on the copy number. Stringent plasmids are present in a limited number (1-2 per cell) while relaxed plasmids occur in large number in each cell.

F-plasmids possess genes for their own transfer from one cell to another, while R-plasmids carry genes resistance to antibiotics. In general, the conjugative plasmids are large, show stringent control of DNA replication, and are present in low numbers. On the other hand, non- conjugative plasmids are small, show relaxed control of DNA replication, and are present in high numbers.

Nomenclature of plasmids:

It is a common practice to designate plasmid by a lower case p, followed by the first letter(s) of researcher(s) names and the numerical number given by the workers. Thus, pBR322 is a plasmid discovered by Bolivar and Rodriguez who designated it as 322. Some plasmids are given names of the places where they are discovered e.g. pUC is plasmid from University of California.

pBR322 — the most common plasmid vector:

pBR322 of E.coli is the most popular and widely used plasmid vector, and is appropriately regarded as the parent or grandparent of several other vectors. pBR322 has a DNA sequence of 4,361 bp. It carries genes resistance for ampicillin (Amp r ) and tetracycline (TeI r ) that serve as markers for the identification of clones carrying plasmids. The plasmid has unique recognition sites for the action of restriction endonucleases such as EcoRI, Hindlll, BamHI, Sail and Pstll (Fig. 6.8).

Other plasmid cloning vectors:

The other plasmids employed as cloning vectors include pUC19 (2,686 bp, with ampicillin resistance gene), and derivatives of pBR322- pBR325, pBR328 and pBR329.

Bacteriophages:

Bacteriophages or simply phages are the viruses that replicate within the bacteria. In case of certain phages, their DNA gets incorporated into the bacterial chromosome and remains there permanently. Phage vectors can accept short fragments of foreign DNA into their genomes. The advantage with phages is that they can take up larger DNA segments than plasmids. Hence phage vectors are preferred for working with genomes of human cells.

Bacteriophage lambda (or simply phage λ), a virus of E.coli, has been most thoroughly studied and developed as a vector. In order to understand how bacteriophage functions as a vector, it is desirable to know its structure and life cycle (Fig. 6.9).

Phage λ consists of a head and a tail (both being proteins) and its shape is comparable to a miniature hypodermic syringe. The DNA, located in the head, is a linear molecule of about 50 kb. At each end of the DNA, there are single-stranded extensions of 12 base length each, which have cohesive (cos) ends.

On attachment with tail to E.coli, phage X injects its DNA into the cell. Inside E.coli, the phage linear DNA cyclizes and gets ligated through cos ends to form a circular DNA. The phage DNA has two fates-lytic cycle and lysogenic cycle.

The circular DNA replicates and it also directs the synthesis of many proteins necessary for the head, tail etc., of the phage. The circular DNA is then cleaved (to form cos ends) and packed into the head of the phage. About 100 phage particles are produced within 20 minutes after the entry of phage into E.coli.

The host cell is then subjected to lysis and the phages are released. Each progeny phage particle can infect a bacterial cell, and produce several hundreds of phages. It is estimated that by repeating the lytic cycle four times, a single phage can cause the death of more than one billion bacterial cells.

If a foreign DNA is spliced into phage DNA, without causing harm to phage genes, the phage will reproduce (replicate the foreign DNA) when it infects bacterial cell. This has been exploited in phage vector employed cloning techniques.

In this case, the phage DNA (instead of independently replicating) becomes integrated into the E.coli chromosome and replicates along with the host genome. No phage particles are synthesized in this pathway.

Use of phage λ as a vector:

Only about 50% of phage λ DNA is necessary for its multiplication and other functions. Thus, as much as 50% (i.e. up to 25kb) of the phage DNA can be replaced by a donor DNA for use in cloning experiments. However, several restriction sites are present on phage λ which is not by itself a suitable vector. The λ-based phage vectors are modifications of the natural phage with much reduced number of restriction sites.

They have just one unique cleavage site, which can be cleaved, and a foreign DNA ligated in. It is essential that sufficient DNA (about 25%) has to be deleted from the vector to make space for the foreign DNA (about 18kb).

These vectors have a pair of restriction sites to remove the non-essential DNA (stuffer DNA) that will be replaced by a foreign DNA. Replacement vectors can accommodate up to 24kb, and propagate them. Many phage vector derivations (insertion/ replacement) have been produced by researchers for use in recombinant DNA technology.

Phage M13 vectors:

Phage M13 (bacteriophage M13) is a single- stranded DNA phage of E.coli. Inside the host cell, M13 synthesizes the complementary strand to form a double-stranded DNA (replicative form DNA RF DNA). For use as a vector, RF DNA is isolated and a foreign DNA can be inserted on it. This is then returned to the host cell as a plasmid. Single- stranded DNAs are recovered from the phage particles. Phage M13 is useful for sequencing DNA through Sanger’s method

Cosmids:

Cosmids are the vectors possessing the characteristics of both plasmid and bacteriophage λ. Cosmids can be constructed by adding a fragment of phage λ DNA including cos site, to plasmids. A foreign DNA (about 40 kb) can be inserted into cosmid DNA.

The recombinant DNA so formed can be packed as phages and injected into E.coli (Fig. 6.10). Once inside the host cell, cosmids behave just like plasmids and replicate. The advantage with cosmids is that they can carry larger fragments of foreign DNA compared to plasmids.

Phasids are the combination of plasmid and phage and can function as either one (i.e as plasmid or phage). Phasids possess functional origins of replication of both plasmid and phage λ, and therefore can be propagated (as plasmid or phage) in appropriate E.coli. The vectors phasids may be used in many ways in cloning experiments.

Artificial Chromosome Vectors:

Human artificial chromosome (HAC):

Developed in 1997 (by H. Willard), human artificial chromosome is a synthetically produced vector DNA, possessing the characteristics of human chromosome. HAC may be considered as a self-replicating micro-chromosome with a size ranging from 1/10th to 1/5th of a human chromosome. The advantage with HAC is that it can carry human genes that are too long. Further, HAC can carry genes to be introduced into the cells in gene therapy.

Yeast artificial chromosomes (YACs):

Introduced in 1987 (by M. Olson), yeast artificial chromosome (YAC) is a synthetic DNA that can accept large fragments of foreign DNA (particularly human DNA). It is thus possible to clone large DNA pieces by using YAC. YACs are the most sophisticated yeast vectors, and represent the largest capacity vectors available. They possess centromeric and telomeric regions, and therefore the recombinant DNA can be maintained like a yeast chromosome.

Bacterial artificial chromosomes (BACs):

The construction of BACs is based on one F-plasmid which is larger than the other plasmids used as cloning vectors. BACs can accept DNA inserts of around 300 kb. The advantage with bacterial artificial chromosome is that the instability problems of YACs can be avoided. In fact, a major part of the sequencing of human genome has been accomplished by using a library of BAC recombinant.

Shuttle Vectors:

The plasmid vectors that are specifically designed to replicate in two different hosts (say in E.coli and Streptomyces sp) are referred to as shuttle vectors. The origins of replication for two hosts are combined in one plasmid. Therefore, any foreign DNA fragment introduced into the vector can be expressed in either host. Further, shuttle vectors can be grown in one host and then shifted to another host (hence the name shuttle). A good number of eukaryotic vectors are shuttle vectors.

Choice of a Vector:

Among the several factors, the size of the foreign DNA is very important in the choice of vectors. The efficiency of this process in often crucial for determining the success of cloning. The size of DNA insert that can be accepted by different vectors is shown in Table 6.5.

Gene Cloning Strategies:

A clone refers to a group of organisms, cells, molecules or other objects, arising from a single individual. Clone and colony are almost synonymous. Gene cloning strategies in relation to recombinant DNA technology broadly involve the following aspects (Fig. 6.13).

a. Generation of desired DNA fragments.

b. Insertion of these fragments into a cloning vector.

c. Introduction of the vectors into host cells.

d. Selection or screening of the recipient cells for the recombinant DNA molecules.

Cloning From Genomic DNA or MRNA?

DNA represents the complete genetic material of an organism which is referred to as genome. Theoretically speaking, cloning from genomic DNA is supposed to be ideal. But the DNA contains non- coding sequences (introns), control regions and repetitive sequences. This complicates the cloning strategies hence DNA as a source material is not preferred, by many workers. However, if the objective of cloning is to elucidate the control of gene expression, then genomic DNA has to be invariably used in cloning.

The use of mRNA in cloning is preferred for the following reasons:

a. mRNA represents the actual genetic information being expressed.

b. Selection and isolation mRNA is easy.

c. As introns are removed during processing, mRNA reflects the coding sequence of the gene.

d. The synthesis of recombinant protein is easy with mRNA cloning.

Besides the direct use of genomic DNA or mRNA, it is possible to synthesize DNA in the laboratory and use it in cloning experiments. This approach is useful if the gene sequence is short and the complete sequence of amino acids is known.

The different strategies for the cloning of genomic DNA and mRNA are described under gene libraries

Genetic Engineering Guidelines:

With the success of Boyer-Cohen experiments (in 1973), it was realised that recombinant DNA technology could be used to create organisms with novel genes. This created worldwide commotion (among scientists, public and government officials) about the safety, ethics and unforeseen consequences of genetic manipulations. Some of the phrases quoted in media in those days are given.

d. The most threatening scientific research.

It was feared that some new organisms, created inadvertently or deliberately for warfare, would cause epidemics and environmental catastrophes. Due to the fears of the dangerous consequences, a cautious approach on recombinant DNA experiments was suggested.

In 1974, a group of ten scientists led by Paul Berg wrote a letter that simultaneously appeared in the prestigious journals-Nature, Science and Proceedings of the National Academy of Sciences.

The dangers of DNA technology were printed out in that letter (highlights given below):

“Recent advances in techniques for isolation and rejoining of segments of DNA now permit construction of biologically active recombinant DNA molecules in vitro. Although such experiments are likely to facilitate the solution of important theoretical and practical biological problems, they would also result in creation of novel types of DNA elements whose biological properties cannot be completely predicted. There is a serious concern that some of these DNA molecules could prove biologically hazardous”.

The letter also appealed to molecular biologists worldwide for a moratorium on many kinds of recombinant DNA research, particularly those involving pathogenic organisms.

Asilomar Recommendations:

In February 1975, a group of 139 scientists from 17 countries held a conference at Asilomar, a conference center in California, USA. They assured the uneasy public that the microorganisms used in DNA experiments were specifically bred and could not survive outside the laboratory. These scientists formulated guidelines and recommendations for conducting experiments in genetic engineering.

NIH Guidelines:

National Institute of Health (NIH), USA constituted the Recombinant DNA Advisory Committee (RAC) which issued a set of stringent guidelines to conduct research on DNA. RAC was in fact overseeing the research projects involving gene splicing and recombinant DNA.

Some of the important original NIH recommendations on recombinant DNA research relate to the following aspects:

a. Physical (laboratory) containment levels for conducting experiments.

b. Biological containment-the host into which foreign DNA is inserted should not proliferate outside the laboratory or transfer its DNA into other organisms.

c. For research on pathogenic organisms, elaborate, controlled and self-contained rooms were recommended.

d. For research on less dangerous organisms, units equipped with high quality filter systems should be used.

e. No deliberate release of any organism containing recombinant DNA into the environment.

It may be noted here that although the NIH guidelines did not have the legal status, most institutions, companies and scientists voluntarily complied.

Relaxation of NIH guidelines:

It was in 1980, the original NIH guidelines were considerably relaxed by NIH-RAC, based on the experience and experimental data obtained from the NIH-sponsored studies on recombinant DNA research. It was almost agreed that the original apprehensions on recombinant DNA research were unfounded.

It is a fact that the genetic engineering research flourished and progressed rapidly after relaxation of NIH guidelines. It may however be noted that NIH- RAC continues to be a watchdog over the DNA technology experiments.

Pharmaceutical products of recombinant DNA:

As the recombinant DNA technology progressed, many pharmaceutical compounds of human health care are being produced through genetic manipulations. Most countries consider that the existing regulations for approval of pharmaceuticals of commercial use are adequate to ensure safety since the process by which the product manufactured is irrelevant. Thus, the recombinant DNA product (protein, vaccine, and drug) is evaluated for its safety and efficacy like any other pharmaceutical product.

Genetically engineered organisms (GEOs):

Recombinant DNA research has resulted in the creation of many genetically engineered organisms. These include microorganisms, animals and plants. The latter two respectively result in transgenic animals and transgenic plants

The Future of Genetic Engineering:

DNA technology has largely helped scientists to understand the structure, function and regulation of genes. The development of new/modern biotechnology is primarily based on the success of DNA technology. Thus, the present biotechnology (more appropriately molecular biotechnology) has its main roots in molecular biology.

Biotechnology is an interdisciplinary approach for applications to human health, agriculture, industry and environment. The major objective of biotechnology is to solve problems associated with human health, food production, energy production and environmental control.

The major contributions of genetic engineering through the new discipline biotechnology are given in this book.

It is an accepted fact that the recombinant DNA technology has entered the main stream of human life and has become one of the most significant applications of scientific research. Biotechnology is regarded as more an art than a science. After the successful sequencing of human genome, many breakthroughs in biotechnology are expected in future.


Which pair of enzymes produce compatible ends?

However, some produce blunt ends. DNA ligase is a DNA-joining enzyme. If two pieces of DNA have matching ends, ligase can link them to form a single, unbroken molecule of DNA.

Similarly, what is the difference between sticky ends and blunt ends? Blunt and sticky ends areresult of restriction endonuclease action on double stranded DNA. Sticky Ends &ndash are staggered ends on a DNA molecule with short, single-stranded overhangs. Blunt Ends are a straight cut, down through the DNA that results in a flat pair of bases on the ends of the DNA.

Additionally, what are cohesive ends?

Longer overhangs are called cohesive ends or sticky ends. They are most often created by restriction endonucleases when they cut DNA. Very often they cut the two DNA strands four base pairs from each other, creating a four-base 5' overhang in one molecule and a complementary 5' overhang in the other.

5' overhang- Restriction enzymes that cleave the DNA asymmetrically leave several single stranded bases. If the single-stranded bases end with a 5' phosphate, the enzyme is said to leave a 5' overhang. If the single-stranded bases end with a 3' hydroxyl, the enzyme is said to leave a 3' overhang.


PCR cloning strategies

PCR cloning is a method in which double-stranded DNA fragments amplified by PCR are ligated directly into a vector. PCR cloning offers some advantages over traditional cloning which relies on digesting double-stranded DNA inserts with restriction enzymes to create compatible ends, purifying and isolating sufficient amounts, and ligating into a similarly treated vector of choice (see insert preparation).

With PCR amplification, this cloning technique requires much less starting template materials which include cDNA, genomic DNA, or another insert-carrying plasmid (see subcloning basics). Furthermore, PCR cloning provides a simpler workflow by circumventing the requirement of suitably-located restriction sites and their compatibility between the vector and insert. Nevertheless, there are a number of considerations related to: PCR primers and amplification conditions, the cloning method of choice and the cloning vectors used, and, finally, confirmation of successful cloning and transformation.

With respect to PCR amplification of a sequence of interest, primers must be designed and PCR conditions (components and cycling) optimized for efficient and specific amplification of the template. Primer design tools are available to bioinformatically evaluate and select suitable target-specific primer sequences for amplification. Ligation requires that either the insert or vector has 5′-phosphorylated termini therefore, if the cloning vector lacks 5′-phosphorylated ends, 5′-phosphate groups must be added to the PCR primers during synthesis or by T4 polynucleotide kinase for successful ligation. For PCR optimization, reaction component concentrations, annealing temperatures, and template amounts are of importance.

TA cloning and blunt-end cloning represent two of the simplest PCR cloning methods. Their choice depends upon the nature of the vector and the type of PCR enzymes used in cloning. TA cloning employs a thermostable Taq DNA polymerase capable of amplifying short DNA sequences. This enzyme lacks 3′→ 5′ proofreading activity and features a terminal transferase activity that adds an extra deoxyadenine at the 3′ end of the amplicons (3′ dA). The resulting PCR products with 3′ dA overhangs are readily cloned into a linearized TA cloning vector containing complementary 3′ deoxythymine (3′ dT) overhangs (Figure 1). While relatively straightforward, the limitations of this method include the length of insert (up to 5 kb), the inability to clone inserts directionally, and the high error rate associated with Taq DNA polymerase.

Blunt-end cloning involves the ligation of an insert into a linearized vector where both DNA fragments lack overhangs. Blunt-end inserts can be produced using high-fidelity DNA polymerases with 3′→5′ exonuclease or proofreading activity. Their proofreading activity improves the sequence accuracy of the amplified products however, limitations include lower ligation efficiencies when inserting into blunt-end cloning vectors and the inability to clone directionally. Ligation efficiency can be improved by incubating the amplicons with a Taq DNA polymerase and dATP in a procedure called “3′ dA tailing” (incubate 20–30 minutes at 72°C), then purifying the 3′ dA-tailed products (Figure 1).

Figure 1. Common PCR cloning strategies.

To further simplify and streamline the cloning workflow, specialized vectors have been developed to place an insert into vector, for example, without using a ligase. One such class of vectors includes the Invitrogen TOPO cloning vectors which contain covalently linked DNA topoisomerase I that functions as both a restriction enzyme and a ligase (learn more about TOPO cloning technology). Compared to conventional PCR cloning vectors, these vectors result in shorter ligation reaction times (e.g., 5 minutes) and greater cloning efficiencies (e.g., >95% positive clones) and with a much simpler protocol. Furthermore, directional cloning of the PCR products can be achieved with a specially designed TOPO vector using a specific primer design.

Regardless of the cloning method choice, cloning efficiencies are significantly improved by purification of PCR amplicons prior to the ligation reaction. PCR clean-up helps remove salts, nucleotides, nonspecific amplicons, and primer-dimers. After ligation and transformation into the appropriate competent cells, the resulting colonies need to be screened carefully for the correct insert, as well as its proper frame and orientation for subsequent studies to analyze gene fusions and/or protein expression.


Finding the Best Ligation Biology

Some recombinant proteins aren’t well tolerated by E. coli and can bring about poor transformation or little colonies. Many proteins have a certain affinity for unique phases of substance mobility. MMR proteins act to stop such recombination. Most enzymes arrive in glycerol solution for a storage buffer, but enzymes don’t do the job well in the existence of high glycerol concentration. Restriction enzymes have to be carefully chosen and may be used to verify the size and orientation of the insert. http://www.bleupiscine.fr/what-is-so-fascinating-about-biology-corner/ Hence, it’s essential to use the very same restriction enzyme for the two sources of DNA to create the matching ligating fragments.

1 method utilized for transfecting cells in cell culture is known as electroporation. Competent cells are commercially readily available for efficient and dependable transformation. All the cells within this colony are identical clones and carry exactly the same recombinant plasmid.

Generally speaking, a greater reaction temperature requires less time but might create a decrease yield. Moreover, there’s a gradient farther down the body, with a tall point at the head end. Viral vectors may More Bonuses also be utilized to transfect eukaryotic cells. As a consequence of cleavage by these enzymes, DNA fragments are made with various sorts of ends like sticky ends and blunt ends.

You don’t wish to be cutting your plasmid in necessary regions like the ORI. So every plasmid containing your intended gene won’t be killed by antibiotics. Restriction endonucleases have the ability to cleave dsDNA and produce DNA fragments with distinctive ends.

To cut DNA at known locations, researchers utilize restriction enzymes that were purified from several bacterial species, and that can be purchased from several industrial sources. Singlecell biology is thought to be a new approach to recognize and validate diseasespecific biomarkers. It’s exciting that singlecell biology could be a crucial approach to spot and validate diseasespecific biomarkers. Abstract Singlecell biology is regarded as a new approach to recognize and validate diseasespecific biomarkers. In the event the DNA that’s introduced comes from a different species, the host organism is currently deemed to be transgenic. RNA aussieessaywriter.com.au/ might also be ligated similarly. PCR may also be utilized to facilitate mutagenesis.

The perfect gas law is an superb illustration of a model, concerning both their usefulness and imperfections. If orientation of an insert is essential, two distinct ends increase the probability of the right orientation. Polarity is a moderately complicated notion. Polarity in the creation of the embryo might be illustrated by the early evolution of the nematode embryo. Thus, whatever results in the asymmetry of vacuole pH also has to be asymmetric between mother and daughter cells ahead of cytokinesis.

The DNA ligation kit comprises the reagents necessary to raise the consistency of ligations. For example, you can use PCR product with A end utilizing taq for ligation, and do the adaptor ligation. If it’s not feasible to use two unique websites, then the vector DNA may want to get dephosphorylated to steer clear of a high background of recircularized vector DNA free of insert. When it’s not feasible to use two unique websites, then the vector DNA may want to get dephosphorylated to steer clear of a high background of recircularized vector DNA free of insert. On both sides of the gene is an subject of DNA known as the sticky end. The MCS, if available, is frequently the first selection for insertion, since the region is made specifically for cloning. In addition, it comprises a colonies counter.

Colony number is nearly identical and low (around 100 per plate) whatever the presence or lack of oligos. It appears to be another illustration of asymmetry. A nice instance of polarity featuring all the necessary classical features is a easy organism, hydra. These ends are called sticky or overhanging ends. Both distinct ends can stop the religation of the vector with no insert, and in addition, it makes it possible for the fragment to be inserted in a directional way. The joined ends might be from a single DNA molecule or from various molecules. In the event the sticky ends on each side of the vector are compatible with one another, the vector is a lot more likely to ligate to itself rather than to the desired insert.

Many blunt-ended ligations are performed at 14-25 C overnight. The magnitude of polarization at any point is dependent on the job of the sun, so there’s a pattern of polarization of the sky for any specific position of the sun. Critical features of ligation reactions are discussed, including how the length of a sticky end overhang impacts the reaction temperature and the way the proportion of DNA insert to vector needs to be tailored to stop self-ligation.


CBSE Class 12 Biology Solved Question Paper 2018

Differentiate between Parthenocarpy and Parthenogenesis. Give one example of each.

In most plants, flowers need to be pollinated and fertilized to produce fruits. However, some plants can produce fruits before fertilization or without fertilization. Parthenocarpy is the process which produces fruits from unfertilized ovules in plants. Unfertilized ovules develop into fruits prior to fertilization. These fruits do not contain seeds.

Parthenogenesis is a type of reproduction commonly shown in organisms mainly by some invertebrates and lower plants. It can be described as a process in which unfertilized ovum develops into an individual (virgin birth) without fertilization. Therefore, it can be considered as a method of asexual reproduction.

It is seen in organisms like rotifers, honeybees and even some lizards and birds (turkey). The key difference between parthenogenesis and parthenocarpy is, parthenogenesis is shown by animals and plants while pathenocarpy is shown only by plants.

Give an example of a bacterium, a fungus and an insect that are used as biocontrol agents.

The bacterium, a fungus and insects that are used as biocontrol agents are:

Insects = Ladybird and Dragonflies.

Bacteria = Bacillus thuringiensis

Looking at the deteriorating air quality because of air pollution in many cities of the country, the citizens are very much worried and concerned about their health. The doctors have declared a health emergency in the cities where the air quality is very severely poor.

(a) Mention any two major causes of air pollution.

(b) Write the two harmful effects of air pollution on plants and humans.

(c) As a captain of your school Eco-club, suggest any two programmes you would plan to organise in the school so as to bring awareness among the students on how to check air pollution in and around the school

a)Two causes of air pollution

(1) The burning of fossil fuels.
(2) Smoke released from vehicles.
(3) Industrial effluents
(4) Smoke stacks of thermal power plants.

(b) Harmful effects of air pollution.
(1) It affects respiratory system of humans and of animals.
(2) It also reduces growth and yield of crops & causes premature death of plants.

(c)
(1) Encouraging public transport i.e. buses & using CNG instead of diesel.
(2) Planting more trees to curb pollution.

Medically it is advised to all young mothers that breastfeedings is the best for their newborn babies. Do you agree? Give reasons in support of your answer.

Yes, i do agree with the fact that breastfeeding is the best for newborn babies.

Mammary glands start producing milk at the end of pregnancy. The milk produced during the initial few days and lactation is called COLOSTRUM which contains several antibodies.

It helps in developing resistance for a newborn baby. It helps the baby fight of viruses and bacteria. Thus breast milk is packed with a disease-fighting substance that protects your baby from illness.

Breast milk also naturally contains many of the vitamins and minerals that a newborn requires. Also, it is easily digested - no constipation, diarrhoea and upset stomach.

Name the most commonly used bioreactor and describe its working.

The most commonly used bioreactors are of stirring type. A stirred - tank reactors is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system. pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

How has the development of bioreactor helped in biotechnology?

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-100 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw material are biologically converted into specific products, individual enzymes, etc., using the microbial plant, animal or human cells.

Expand VNTR and describe its role in DNA fingerprinting.

VNTR stands for &ldquoVariable Number of Tandem Repeats&rdquo.
The VNTR belongs to a class of satellite DNA referred to as mini-satellite. A small DNA sequence is arranged tandemly in many copy numbers. The copy number varies from chromosome to chromosome in an individual. The numbers of repeat show very high degree of polymorphism. As a result th size of VNTR varies in size from 0.1 to 20 kb. Consequently, after hybridization with VNTR probe, the autoradiogram gives many bands of differing sizes. These bands give characteristic pattern for an individual DNA which is used to identify individuals.

Draw a diagram of a mature human sperm. Label any three parts and write their functions.

(1) Acrosome: It is a cap-like structure, filled with hydrolytic enzymes that help fertilisation of the ovum.

(2) Middle piece: Possesses numerous mitochondria, which produces energy for the movement of tail.

(3) Tail: Facilitate sperm motility essential for fertilisation.

Explain the roles of the following with the help of an example each in recombinant DNA technology:

  1. Restriction enzymes belong to the class of enzymes nucleases which breaks nucleic acids by cleaving their phosphodiester bonds.
  2. Since Restriction endonucleases cut DNA at specific recognition site, they are used to cut the donor DNA to isolate the desired gene.
  3. The desired gene has sticky ends which can be easily ligated to cloning vector cut by same restriction enzymes having complementary sticky ends to form recombinant DNA
  4. An example is EcoR1 which is obtained from E.coli bacteria &ldquoR&rdquo strain which cuts DNA at specific palindromic Recognition site.
    5&lsquo GAATTC 3&lsquo
    3&lsquo CTTAAG 5&lsquo
  1. Plasmids are autonomous, extrachromosomal circular double-stranded DNA of bacteria
  2. Since they are small and self replicating,they are used as cloning vectors in genetic engineering.
  3. Some plasmids have antibiotic resistance genes which can be used as marker genes to identify recombinant plasmids from non-recombinant ones.
  4. The plasmids are cut and ligated with desired genes and transformed into a host cell for amplification to obtain the desired products.
  5. An example of artificially modified plasmids is pBR322 ( constructed by Bolivar and Rodriguez) or pUC (constructed at university at California).

List any two applications of DNA fingerprinting technique.

Since DNA from every tissue (such as blood, hair - follicle , skin, bone, saliva, sperm etc.), from an individual, show the same degree of polymorphism, they become very useful identification tool in forensic
applications to identify criminals. Further, as the polymorphisms are inheritable from parents to children, DNA fingerprinting is the basic of paternity testing, in case of disputes.


Nonhomologous DNA end-joining (NHEJ) is the predominant double-strand break (DSB) repair pathway throughout the cell cycle and accounts for nearly all DSB repair outside of the S and G2 phases. NHEJ relies on Ku to thread onto DNA termini and thereby improve the affinity of the NHEJ enzymatic components consisting of polymerases (Pol μ and Pol λ), a nuclease (the Artemis·DNA-PKcs complex), and a ligase (XLF·XRCC4·Lig4 complex). Each of the enzymatic components is distinctive for its versatility in acting on diverse incompatible DNA end configurations coupled with a flexibility in loading order, resulting in many possible junctional outcomes from one DSB. DNA ends can either be directly ligated or, if the ends are incompatible, processed until a ligatable configuration is achieved that is often stabilized by up to 4 bp of terminal microhomology. Processing of DNA ends results in nucleotide loss or addition, explaining why DSBs repaired by NHEJ are rarely restored to their original DNA sequence. Thus, NHEJ is a single pathway with multiple enzymes at its disposal to repair DSBs, resulting in a diversity of repair outcomes.

This work was supported by National Institutes of Health grants (to M. R. L.). This is the second article in the Thematic Minireview series “DNA double-strand break repair and pathway choice.” The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


Transfection

Transfection is same as transformation but here instead of plasmid, phage DNA is involved. Here also like plasmid DNA the purified phage DNA, or recombinant phage molecule is mixed with competent E.coli cells and DNA uptake is induced by a heat shock treatment.

V isualization of phage infection

Cell lysis is the final stage of phage infection. Immediately after transfection with phage DNA, when infected cells are spread on to a solid agar medium then cell lysis can be visualized as plaques on a lawn of bacteria (Figure-20). Each plaque is a zone of clearing produced as the phage lyse the cells and move on to infect and eventually lyse the neighbouring bacteria. Plaques are formed by both &lambda phage and M13 phage. True plaques are formed by &lambda whereas M13 does not form true plaques as &lambda phage, because it doesn't lyse the host cell. M13 instead causes a decrease in the growth rate of infected cells sufficient to produce a zone of relative clearing on a bacterial lawn.

Figure 20: Formation of plaques on a lawn of bacteria

The end result of a gene cloning experiment using a &lambda or M13 vector is therefore an agar plate covered in phage plaques. Each plaque is derived from a single transfected or infected cell and therefore contains identical phage particles.

M13 phage vectors

The filamentous phage partcles contain a 6.7kb circular single stranded DNA. After infection of a sensitive E.coli host, the complementary strand is synthesized, and the double stranded DNA is known as replicative form(RF) with about 100 copies per cell. The cells are not lysed by M13, but continues to grow slowly, and single stranded forms are continuously packaged and released from the cells as new phage particles. The same single strand of the complementary pair is always present in the phage particle.

The useful properties of M13 as a vector are that the RF can be purified and manipulated exactly like a plasmid, but the same DNA may be isolated in a single-stranded form from phage particles in the medium.


Negative controls in cloning? - (Aug/16/2005 )

I use the double-digest method to cut the PCR product and the plasmid. Then I will do the ligation. So, what kinds of negative controls should be used in the ligation steps?

Somebody told me as the following, but I can not understand. so, waht kind of negative control do you usually uased?

Negative Controls:
Reaction without insert -> if colonies then backbone is not completely double
cut
Reaction without insert and ligase -> if colonies then backbone is not completely single
cut
Reaction without template and ligase -> if colonies then insert is contaminated with PCR
template

Those are the three major controls, but we usually only use the first one.

If it helps you understand, think about what's in your ligation reaction, where each component came from, and what the possible contaminants are. Positive colonies will arise from any plasmid containing a gene for drug resistance. These plasmids can be one of three things.

1) Your ligated construct - your insert ligates to your vector as you expect, giving rise to a drug resistant bacteria, and therefore a colony.

2) Self-ligated vector without insert - the cut vector ligates its two ends together without an insert. This can happen if you're working with blunt-end ligation, your sticky ends have partial overlap in sequence complementarity, or if there are trace amounts of contaminating nuclease that cause your vector's ends to become blunt. This type of potential contaminant is the reason the "no insert" control is run. The control also addresses the possibility that your vector wasn't fully cut - if you don't actually remove the small piece of DNA between your cloning sites, the vector will be a linear piece of DNA with complementary ends. You can minimize the problem by treating vector (NOT the insert) with phosphatase prior to gel extraction.

3) "Source" vectors - in a ligation reaction, you combine two different pieces of DNA with complementary ends. Each of those pieces of DNA came from somewhere - the vector from a full-length plasmid and the insert from either a full-length plasmid or a PCR amplification of genomic DNA or a plasmid. Whatever the case, those source materials are present until you remove them, which is usually accomplished by agarose gel extraction - you separate your components based on size and specifically extract the productive ligation material away from the source material. However, if your source DNA runs at similar positions to your ligation material, you may wind up co-purifying them, and they'll get included in your transformation. This is why the "no ligase" controls are done - to make sure that the purified ligation materials aren't carrying any source vectors.

so, what kind of negative controls do I really need in my experiment? please tell me the detail cocktail, thank you!

hi
i do this negative control : mix ligase bufer water and enzyme divide it in two and add in the first one plasmid + insert and in the second one plasmid + water.
fred

one tube with liagtion of insert and double-digest plasmid and another tube only with double-digest plasmid


Watch the video: Blunt ends VS sticky ends. (December 2021).