FEDERAL UNIVERSITY NDUFU-ALIKE IKWOP.M.B 1010 ABAKALIKI, EBONYI STATE, NIGERIA

FACULTY OF SCIENCE AND TECHNOLOGYCOURSE CODE: BTG 202 COURSE TITLE: MOLECULAR GENETICS I (3 CREDITS)

2ND SEMESTER 2016/2017 SESSION

DNA-Basics of Structure and Analysis.

The following diagram displays the flow of genetic information from DNA to the protein. This can be interpreted in genetic terms by saying that information contained in genes (DNA) is eventually expressed as the phenotype (protein).

The organelles of plants are both of a prokaryotic (chloroplast and mitochondria) and eukaryotic (nucleus) origin. The organelles contain DNA as well as the nucleus. Also, found within each organelle are all of the steps of the central dogma of molecular genetics. Reverse transcription has not been demonstrated in the organelles, but plant mitochondria undergo mRNA editing, a feature not known for nuclear expressed genes. These functions are:

1. Enzymatic2. Structural components3. Regulatory roles

Original Concept of the Gene: One gene = one enzyme

This concept does not hold for those proteins that are heteromeric or more different subunits.

For instance: RUBISCO (ribulose bisphosphate carboxylase oxygenase)

CO2 + 5-C-sugar --------------------> 2 3-C-sugars

RUBISCO is a multimeric protein of 16 peptides

8 small subunits (nuclear encoded)

8 large subunits (chloroplast encoded)o --thus two genes are responsible for this enzyme

Revised Concept: One gene = one peptide

Recently Glucose 6-phosphate dehydrogenase is discovered. It is an enzyme that can be seen in human red blood cells. The real nature of this peptide is encoded in sequence from two chromosomes.

Minor form: NH4--------------------CO3 (The information encoding the genetic material are on the X chromosome.)

Major form: NH4xx------------------CO3 (Amino acids 1-53 are encoded on chromosome 6, and amino acids 54-479 are encoded on the X chromosome)

Summary: Genetic material fits the one gene = one peptide replica affirmed above, if you be ready to accept that not every genes exist in only constant stretch of gene.

REASONS WHY DNA IS A GENETIC MATERIAL

Some researchers has done a lot of work to confirm that DNA is a Genetic Material among them are:

(1) Griffith: As a researcher, he added dead, potent pneumonia microorganisms to a mouse, it lived; but if he added dead active microorganisms to exist non-virulent microorganisms several rats died. He termed the substance that altered the non-virulent bacteria to virulent the transforming theory.

(2) Avery, MacLeod and McCarty: This group researcher used biochemical sanitization of cellular fractions to conclude that genetic material and not RNA or protein was the transforming theory. Transformation – is the change or modification of phenotype by the addition of foreign gene.

(3) Hershey and Chase the "Blender Experiment"

1. They also knew that something from T2 phage entered E. coli cells and directed the bacteria to produce more phage.

2. They assumed that the genetic material was the material that entered the cell.3. They set out to determine the chemical nature of the material.4. By analyzing the products they determined that 32P entered the cell.5. Thus DNA must be the genetic material. DNA Structure

(4) Watson and Crick Model of DNA: The features of the DNA molecule discovered were as described below:

1. 2 chains2. Purine opposite a pyrimidine3. Chains held together by H-bonds

o Guanine is paired with cytosine by three H-bondso Adenine is paired with thymine by two H-bonds

4. Anti-parallel orientation of the two chains

5'--------------->3'3'<---------------5'

5. The molecule is stabilized by:

o H-bondso Hydrophobic bonding between the stacked bases

Components of Genetic material: DNA is composed of two chains of repeating nucleotides. Each nucleotide consists of three components. These components are:

1. Phosphate Group2. 2-deoxyribose sugar3. A nitrogen containing base

Cytosine Adenine Guanine thymine

Types of Genetic material

1. Watson and Crick described the DNA molecule that was in the B form.2. Genetic material can exist in several other forms.3. The main disparity between the forms is the way that the coil spirals.

A, B, C = right-handed helix Z = left-handed helix (establish in vitro in high salt)

4. B is the major form that is found in the cell.5. Z-DNA was initially found only under high salt conditions, but the cellular

environment is actually a low-salt environment. Some features have been revealed that can become stable Z-DNA under or within a low salt environment.

Alternating purine/pyrimidine tractso poly GC or poly AT stretches

5-methyl-cytosine

Both of these conditions can live in the cell, it is recommended that stretches of Z-DNA might really live in the cell along with other stretches of B-DNA.

In addition to the direction the molecule turns, several other differences exist between the various forms of DNA. The table below summarizes the features of the different forms of Genetic Material.

Form Direction Bases/360o Turn

HelixDiameter

A Right 11.0 23AB Right 10.0 19AC Right 9.3 19AZ Left 12.0 18A

The Restriction Modification System (RMS) involves the following:

1. Viruses attack all types of cells.2. Bacteria are one favorite target.3. Microorganism developed Protection mechanisms on or after these invasions.4. The system they possess for this defense is the restriction-modificiation system.5. This system is composed of a restriction endonuclease enzyme and a methylase

enzyme’6. Microbial species and strain has their own grouping of restriction and methylating

enzymes.

Restriction enzyme: – refers to an enzyme that cuts Genetic Material at internal phosphodiester bonds; different types exist and the most useful ones for molecular biology (Type II) which cleave at a specific DNA sequence.

Methylase – refers to an enzyme that adds a methyl group to a molecule; in restriction-modification systems of bacteria a methyl group is added to DNA at a specific site to protect the site from restriction endonuclease cleavage.

A number of different types of restriction: Enzymes have been found but the most useful ones for molecular biology and genetic engineering are the Type II restriction enzymes. These enzymes cut genetic material at specific nucleotide sequences. Example, the enzyme E. coIi recognizes the sequence:

5' - G A A* T T C - 3'3' - C T T *A A G - 5'

Nucleic Acid Hybridizations

The hybridization of a radioactive probe to filter bound DNA or RNA is one of the most informative experiments that are performed in molecular genetics. Two basic types of hybridizations are possible.

Southern hybridization - hybridization of a probe to strain bound genetic material; the DNA is usually transferred to the filter from a gel

Northern hybridization - hybridization of a probe to strain bound RNA; the RNA is normally transferred to the filter from a gel

Probes are instrument used to identify matching sequences of interest. It is a clone developed by inserting genetic material into a vector. Most often these are plasmid clones. Probe – refers to a single-stranded nucleic acid that has been radiolabelled and is used to identify a complimentary nucleic acid sequence that is membrane bound nucleic acid.

The hybridization process involves two different steps:1. The nucleic acid must be immobilized on a filter. This is generally called a

"Southern Transfer" procedure.2. The actual hybridization of the probe to the filter bound nucleic acid.

Steps describe the Southern transfer method:

1. Digest genetic material with the restriction enzyme of choice.2. Fill the absorption onto an agarose gel and apply an electrical current. 3. Genetic material is negatively charged so it migrates toward the positive pole.4. The distance exact portion migrates is inversely relative to the fragment size.5. Stain the gel with a fluorescent dye which intercalates into the DNA molecule.6. Genetic material can be visualized with a Ultra Violet light source to assess the

completeness of the digestion.7. Denature the double-stranded remains by soaking the gel in alkali (>0.4 M

NaOH).8. Move the Genetic material to a filter membrane (nylon) by capillary action.

Southern transfer’s setup contains (from bottom to top):

o buffero spongeo filter papero the gel containing the nucleic acido a nylon or nitrocellulose membraneo more filter papero paper towels to catch the buffer that passed through all of the above

9. Southern hybridizations with plant DNA is not a trivial matter.10.The primary requirement for a successful experiment is that the DNA to be

probed is digested to completion.

If the genetic materials are completely digested and are sure that it was effectively transferred to the filter membrane, the next step is performing the actual hybridization. The following steps describe the method.

Southern Hybridization Methods

1. Arrange a probe by nick translation or random, oligo-primed labelling.2. insert the probe to a filter (nylon or nitrocellulose) to which single-stranded

nucleic acids are bound. (The filter is protected with a pre hybridization solution which contains molecules which fill in the spots on the filter where the nucleic acid has not bound.

3. Hybridize the single-stranded probe to the filter-bound nucleic acid for 24 hr. The probe will bind to complementary sequences.

4. Wash the filter to remove non-specifically bound probe.5. Expose the filter and determine:

o a. Did binding occur?o b. If so, what is the size of hybridizing fragment?

Hybridization Stringency

1. The temperature and salt concentrations at which we perform hybridization have a direct effect upon the results that are obtained. Specifically, you can set the conditions up so that your hybridizations only occur between the probe and a filter bound nucleic acid that is highly homologous to that probe. You can also adjust the conditions of the hybridization to a nucleic acid that has a lower degree of homology to the probe.

2. Your hybridization results are directly related to the number of degrees below the temperature of DNA at which the experiment is performed. For an aqueous solution of DNA (no salt) the formula for Tm is: Tm = 69.3oC + 0.41(% G + C)oC

3. Hybridizations though are always performed with salt. This requires another formula that takes the salt concentration into account. Under salt-containing hybridization conditions, the effective Tm is what controls the degree of homology between the probe and the filter bound DNA is required for successful hybridization.

The formula for the effective temperature (Eff. Tm).

Eff Tm = 81.5 + 16.6(log M [Na+]) + 0.41(%G+C) - 0.72(% form amide)

4. The salt solution that is most often used in hybridization experiments is standard sodium citrate (SSC). Different concentrations of this solution are used at different steps in the hybridization procedure. The following table gives the Na+ concentration for different strengths of SSC. Remember that this value is essential to derive the Effective Tm.

Na+ ion concentration of different strengths of Standard Sodium Citrate (SSC)

SSC Content [Na+] M20X 3.300010X 1.65005X 0.82502X 0.33001X 0.16500.1X 0.0165

Another relevant relationship is that 1% mismatches of two DNAs lowers the Tm 1.4oC. So in a hybridization with wheat germ that is performed at Tm - 20oC (=67.5oC), the two DNAs must be 85.7% homologous for the hybridization to occur. 100% - (20oC/1.4oC) = 85.7% homology

Let's now look at an actual experiment, the hybridization of a probe with filter bound wheat DNA in 5X SSC at 65oC. The first step is to derive the Eff Tm.

Eff Tm = 81.5 + 16.6(log 0.825) + 18.5 = 98.6oC

These types of hybridization experiments are typically performed at Tm - 20oC. A typical temperature of hybridization is 65oC. (If form amide is used the hybridization is normally performed at 42oC). With these conditions, 83.1% homology between the probe and filter bound DNA is required for hybridization. The following calculation is how this number was derived.

100 - [(98.6-65.0)/1.4] = 100 - (23.6/1.4) = 83.1%.

5. The next step in a hybridization experiment is to wash the filter. This is normally done in two steps. First a non-stringent wash is performed to remove the non-specifically bound DNA and the second wash is performed at a higher stringency that only permits highly homologous sequences to remain bound to the filter. Controlling the stringency is an important step in these experiments.

Stringency - a term used in hybridization experiments to denote the degree of homology between the probe and the filter bound nucleic acid; the higher the stringency, the higher percent homology between the probe and filter bound nucleic acid

Non-stringent wash: normally 2X SSC, 65oC

Eff Tm = 81.5 + 16.6[log(0.33)] + 0.41(45%)= 92.0oC

%Homology = 100 - [(92-65)/1.4] = 80.7%

Stringent wash: normally 0.1X SSC, 65oC

Eff Tm = 81.5 + 16.6[log(0.0165)] + 0.41(45%) = 70.4oC

Importance of the Southern Hybridizations

Southern hybridizations have many applications:

First Application

1. The first application after cloning a gene is often to determine how many copies of the gene are in the species from which the gene was cloned.

2. This experiment is performed by hybridizing a clone of the gene to total DNA that has been digested with several enzymes.

3. The procedure is termed a genomic southern.4. One gene that has drawn intense interest because of its potential applied usage

in plant biotechnology is chitins.5. The gene has also been cloned from other species.6. These hybridizations show that these species contain different copy numbers

for the gene.

Second Application for Southern Hybridizations:

1. The estimation of copy number of a specific gene.2. This experiment is performed by running several lanes with different copy

numbers of the gene.3. You are probing and comparing the hybridization intensities with a companion

genomic southern experiment.4. This is called a reconstruction experiment.5. Analysis can also be performed to determine if a phenotypic mutation is due to a

structural change in the gene controlling the trait of interest.6. If a gene undergoes an insertion or deletion the resulting hybridization pattern

would be changed.7. Insertion mutagenesis would generate fragments of an increased size whereas

deletions would reduce the size of the hybridizing band.8. Two tomato mutants, Never ripe (nr) and ripening inhibitor (ri) express

polygalacturonase, an enzyme involved in fruit ripening, at lower levels than normal or wild type tomato.

9. The question posed here was whether the structure of the polygalacturonase (and other ripening specific genes) is structurally different than the wild type genotype.

Phylogenetics is the study of evolutionary relationships among biological entities - often species, individuals or genes (which may be referred to as taxa ). The major elements of phylogenetics are summarised in Figure 1 below. It is the way that biologists reconstruct the pattern of events that have led to the distribution and diversity of life. Introduction

Population genetic structure refers to the geographical pattern of genetic diversity within or among populations. It could be influenced by gene flow, genetic drift, selection, mutation and recombination. Gene flow is caused by the movement of individuals from one population to another. Estimation of the gene flow level allows conservation biologists to understand the relationships between populations and assess levels of genetic variation in order to evaluate the relative levels of conservation concern hierarchically across populations in a species. Genetic drift is the change in the frequency of a gene variant in a population due to random sampling 1Genetic drift may lead to disappearance of gene variants and thereby reduce genetic diversity.

Phylogeography connects historical processes in evolution with spatial distributions. Analysis of mitochondrial data promoted the empirical development of phylogeography. The statistical phylogeography is one of the widely used approaches in phylogeography. The climate associated with Pleistocene glacial cycles in East Asia was likely mild and characterized by a mosaic of mountains. The past climatic events, such as the Quaternary glaciation, are believed to have played an important role in forming the geographical pattern of the montane species and could leave the vestiges in geographical distribution of genetic diversity of population.

Phylogenetic analyses

This has to do with substitution model selection eg:

1. jModelTest 2.1.4, the TIM2+I+G model etc.2. Bayesian inference (BI) can be used to generate a phylogenetic hypothesis of the DNA

haplotypes.3. BI can be used with Mr Bayes 3. 2, with 1,200,000 generations, sampling trees every 100

generations.4. Two independent runs each with four simultaneous Monte Carlo Markov chains

(MCMC) can also be conducted.5. The convergence of chains can be used to confirm average standard deviation of split

frequency of below 0.01 (0.009889).6. To determine the potential scale reduction factor (PSRF) for all parameters.7. In phylogenetic analysis S. boulengeri can also be used as out group.

Population structure occurs based on the statistics and differentiation. It involves:

1. Most values are high and statistically significant.2. Test of the genetic and geographical distance of the populations of plant and animals

existing in particular area.3. Record of results of the genetic material and geographical distance of the population of

plants and animals existing in particular area have to be taken.4. Pattern of genetic divergence Sequencing and the distributions of genetic variations

among the plants and animals.5. Individual mating of animals within the population has to be recorded.6. It involves AMOVA results because genetic variation must occur among populations and

differentiation within the populations.7. A topography and climate change has to be recorded.

8. Genetic differentiation forming genetic structure pattern has to be recorded.9. Topographic features, climatic fluctuation and anthropogenic activity.

Population structure analyses comparisons using

1. Difference as distance method2. The partition of genetic diversity within and among populations.3. Analysis of molecular variance (AMOVA).4. To assess the significance of isolation.5. Random permutations on matrices of population values.6. The geographical distances can be estimated using eg: Arlequin3.5.1.2.7. Geographical distances between populations can be calculated.8. The spatial genetic structure of haplotypes can be analyzed using the program

SAMOVA1.0 with 1,000 permutations.9. The number of initial conditions can be set to 100 as recommended by researchers..

Differentiation

The cost-effective molecular tool allows the amplification of minute amounts of DNA effectively opened for the field of molecular ecology. These techniques and the advances provide the following:

1. The understanding of sibling species complexes.2. Clonal structure..3. Population structure.4. Phylogeographic patterns.5. Phylogenetic relationships in rotifers.6. Most of the research to date has focused on the rotifer species complex Brachionus

plicatilis.7. The use of DNA sequence and microsatellite variation.8. The background knowledge of life history, mating behaviour, and temporal population

dynamics in these organisms eg: the processes of shaping the genetic diversity in aquatic invertebrates.

9. Rotifers populations with a very high number of clones in genetic equilibrium.10. It ensures that population clonal selection is effective in eroding the number of clones.11. Rotifer populations are strongly differentiated genetically for neutral markers. even at

small12. It differentiates geographical scales, and exhibit deep phylogeographic structure.

Molecular Markers

A molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers are used in molecular biology and biotechnology to identify a particular sequence of DNA in a pool of unknown DNA.

A molecular marker (identified as genetic marker) is a fragment of DNA that is associated with

a certain location within the genome. Molecular markers are used in molecular biology and biotechnology to identify a particular sequence of DNA in a pool of unknown DNA will be necessary.A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter.

Uses of Molecular marker:1. It reveals certain characteristics about the respective source. For example, is a molecular

marker containing information about genetic disorders, genealogy and the evolutionary history of life.

2. Specific regions of the DNA (genetic markers) are used : to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding).3. Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.4. Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's

disease.5. Non-biological molecular markers are also used, for example, in environmental studies.6. Biotechnology to identify a particular sequence of DNA in a pool of unknown DNA of

plant and animals.

Molecular markers are effective because1. They identify an abundance of genetic linkage between identifiable locations within a

chromosome.2. They are able to be repeated for verification.3. They can identify small changes within the mapping population enabling distinction

between a mapping species.4. It allows segregation of traits and identity.5. They identify particular locations on a chromosome.6. It allows physical maps to be created.7. Lastly they can identify how many alleles an organism has for a particular trait (bi allelic

or poly allelic).

List of Markers AcronymRestriction Fragment Length Polymorphism RFLPRandom Amplified Polymorphic DNA RAPDAmplified Fragment Length Polymorphism AFLPVariable Number Tandem Repeat VNTROligonucleotide Polymorphism OPRandom Amplified Polymorphic DNA RAPD

Single Nucleotide Polymorphism SNPAllele Specific Associated Primers ASAPInverse Sequence-tagged Repeats ISTRInter-retrotransposon Amplified Polymorphism IRAP

Cloning Vector

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.A vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.

Features of Vectors:

1. Allow the convenient insertion or removal of DNA fragment in or out of the vector.2. By treating the vector and the foreign DNA with a restriction enzyme.3. That cuts the DNA fragments.4. That contains either blunt ends or overhangs known as sticky ends.5. That can generate Vector DNA and foreign DNA with compatible ends.6. Vectors that can then be joined together by ligation.7. A DNA fragment that has been cloned into a cloning vector.8. A DNA that may be further subcloned into another vector designed for more specific use.

Types of Cloning Vectors that are most commonly used genetic engineers are (i) plasmids. Cloning is generally first performed using Escherichia coli, and cloning vectors in E. coli include plasmids, (ii) bacteriophages (such as phage λ), (iii) cosmids, and (iv) bacterial artificial chromosomes (BACs). Some DNA, however, cannot be stably maintained in E. coli, for example very large DNA fragments, and other organisms such as yeast may be used. Cloning vectors in yeast (v) yeast artificial chromosomes (YACs).

Features of a cloning vector

Commonly used cloning vectors in molecular biology have key features necessary for their function, such as a suitable cloning site and selectable marker. Others may have additional features specific to their use. For reason of ease and convenience, cloning is often performed using E. coli. Thus, the cloning vectors used often have elements necessary for their propagation and maintenance in E. coli, such as a functional origin of replication (ori). The ColE1 origin of replication is found in many plasmids. Some vectors also include elements that allow them to be maintained in another organism in addition to E. coli, and these vectors are called shuttle vector.

Cloning site

All cloning vectors have features that allow a gene to be conveniently inserted into the vector or removed from it. This may be a multiple cloning site (MCS) or polylinker, which contains many unique restriction sites. The restriction sites in the MCS are first cleaved by restriction enzymes, then a PCR-amplified target gene also digested with the same enzymes is ligated into the vectors using DNA ligase. The target DNA sequence can be inserted into the vector in a specific direction if so desired.

Other cloning vectors may use topoisomerase instead of ligase and cloning may be done more rapidly without the need for restriction digest of the vector or insert. In this TOPO cloning method a linearized vector is activated by attaching topoisomerase I to its ends, and this "TOPO-activated" vector may then accept a PCR product by ligating both the 5' ends of the PCR product, releasing the topoisomerase and forming a circular vector in the process. Another method of cloning without the use of DNA digest and ligase is by DNA recombination, for example as used in the Gateway cloning system. The gene, once cloned into the cloning vector (called entry clone in this method), may be conveniently introduced into a variety of expression vectors by recombination.

Selectable marker

A selectable marker is carried by the vector to allow the selection of positively transformed cells. Antibiotic resistance is often used as marker, an example being the beta-lactamase gene, which confers resistance to the penicillin group of beta-lactam antibiotics like ampicillin. Some vectors contain two selectable markers, for example the plasmid pACYC177 has both ampicillin and kanamycin resistance gene. Shuttle vector which is designed to be maintained in two different organisms may also require two selectable markers, although some selectable markers such as resistance to zeocin and hygromycin B are effective in different cell types. Auxotrophic selection markers that allow an auxotrophic organism to grow in minimal growth medium may also be used; examples of these are LEU2 and URA3 which are used with their corresponding auxotrophic strains of yeast.

Another kind of selectable marker allows for the positive selection of plasmid with cloned gene. This may involve the use of a gene lethal to the host cells, such as barnase, Ccda, and the parD/parE toxins. This typically works by disrupting or removing the lethal gene during the cloning process, and unsuccessful clones where the lethal gene still remains intact would kill the host cells, therefore only successful clones are selected.

Reporter gene

Reporter genes are used in some cloning vectors to facilitate the screening of successful clones by using features of these genes that allow successful clone to be easily identified. Such features present in cloning vectors may be the lacZα fragment for α complementation in blue-white selection, and/or marker gene or reporter genes in frame with and flanking the MCS to facilitate the production of fusion proteins. Examples of fusion partners that may be used for screening are the green fluorescent protein (GFP) and luciferase.

Elements for expression

A cloning vector need not contain suitable elements for the expression of a cloned target gene, such as a promoter and ribosomal binding site (RBS), many however do, and may then work as an expression vector. The target DNA may be inserted into a site that is under the control of a particular promoter necessary for the expression of the target gene in the chosen host. Where the promoter is present, the expression of the gene is preferably tightly controlled and inducible so that proteins are only produced when required. Some commonly used promoters are the T7 and lac promoters. The presence of a promoter is necessary when screening techniques such as blue-white selection are used.

Cloning vectors without promoter and RBS for the cloned DNA sequence are sometimes used, for example when cloning genes whose products are toxic to E. coli cells. Promoter and RBS for the cloned DNA sequence are also unnecessary when first making a genomic or cDNA library of clones since the cloned genes are normally subcloned into a more appropriate expression vector if their expression is required.

Some vectors are designed for transcription only with no heterologous protein expressed, for example for in vitro mRNA production. These vectors are called transcription vectors. They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.

Types of cloning vectors

A large number of cloning vectors are available, and choosing the vector may depend on a number of factors, such as the size of the insert, copy number and cloning method. Large insert may not be stably maintained in a general cloning vector, especially for those with a high copy number, therefore cloning large fragments may require more specialized cloning vector.

The pUC plasmid has a high copy number, contains a multiple cloning site (polylinker), a gene for ampicillin antibiotic selection, and can be used for blue-white screen.

Plasmid

Plasmid vector

Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the ones most commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb in size. One of the earliest commonly used cloning vectors is the pBR322 plasmid. Other cloning vectors include the pUC series of plasmids, and a large number of different cloning plasmid vectors are available. Many plasmids have high copy number, for example pUC19 which has a copy number of 500-700 copies per cell, and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.

Some plasmids contain an M13 bacteriophage origin of replication and may be used to generate single-stranded DNA. These are called phagemid, and examples are the pBluescript series of cloning vectors.

Bacteriophage

The bacteriophages used for cloning are the phage λ and M13 phage. There is an upper limit on the amount of DNA that can be packed into a phage (a maximum of 53 kb), therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors may need to have some non-essential genes deleted, for example the genes for lysogeny since using phage λ as a cloning vector involves only the lytic cycle.There are two kinds of λ phage vectors - insertion vector and replacement vector. Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted. In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.

There is also a lower size limit for DNA that can be packed into a phage, and vector DNA that is too small cannot be properly packaged into the phage. This property can be used for selection - vector without insert may be too small, therefore only vectors with insert may be selected for propagation.

Cosmid

Cosmids are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (cos) which contains elements required for packaging DNA into λ particles. It is normally used to clone large DNA fragments between 28 and 45 Kb.

Bacterial artificial chromosome

Insert size of up to 350 kb can be cloned in bacterial artificial chromosome (BAC). BACs are maintained in E. coli with a copy number of only 1 per cell.[16] BACs are based on F plasmid, another artificial chromosome called the PAC is based on the P1 phage.

Yeast artificial chromosome

Insert of up to 3,000 kb may be carried by yeast artificial chromosome.

Human artificial chromosome

Human artificial chromosome may be potentially useful as a gene transfer vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function. It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.

An LB agar plate showing the result of a blue white screen. White colonies may contain an insert in the plasmid it carries, while the blue ones are unsuccessful clones.

Screening: example of the blue/white screen

Blue white screen

Many general purpose vectors such as pUC19 usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for E. coli β-galactosidase, whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based lacZα sequence of the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.