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a thousand ills require a thousand cures Ovid (43 BCE17 CE)

The replication of DNA is not a perfect process, despite many safeguards against mistakes. Although infrequent, errors that affect one or more base pairs can occur. External agents, including various chemicals, ultraviolet light, radiation, and radioactive compounds can permanently alter the sequence of nucleotides of a DNA molecule.

A change to the genetic material is called a mutation. An agent that induces mutations is a mutagen. In the absence of any evidence of a mutagenic effect, a naturally occurring mutation is considered to be spontaneous

In 1927 H. J. Muller showed for the first time that mutations could be induced in Drosophila by use of external agents or mutagens. He also found that increasing the dose of X-rays results in a linear increase in the frequency of mutations. By 1930s, it became established that physical agents such as X-rays, gamma rays, UV radiation, and some chemical agents are all effective as mutagens.

Radiation breaks chromosomes by a chemical reaction which requires energy. The mutagenic effect of radiation depends upon the amount and type of energy left in the tissue. Visible light is a less energetic radiation as it leaves energy in the form of heat. The energetic radiations such as ultraviolet (UV) leave energy in the form of heat and activation which leads to chemical change.

Due to their shorter wavelength, X-rays and gamma rays penetrate tissues deeper than visible and UV light. They can impart enough localised energy to absorbing tissue to ionise atoms and molecules tn the process called ionisation. Ions produce breaks in chromosomes (DNA) thereby inducing mutations. The free ions may combine with oxygen and produce highly reactive chemicals which may also react with DNA and cause mutagenesis.

When a hydrogen atom consisting of one proton and one electron is ionised, the free electron may directly interact with DNA. Or the electron may interact with a molecule of water to produce OH, a free radical which can cause damage to DNA in the same way as the free electron.

E. Altenberg was first to show that ultraviolet radiation can induce mutations. UV rays have longer wavelengths than X-rays and gamma rays, hence they cannot penetrate tissues as deeply. Their mutagenic action is limited to bacteria, fungal spores or other free cells whose genetic material lies very near the irradiated surface.

Alkylating Agents is the most powerful group of mutagens. They transfer alkyl groups to the nitrogen atoms of the bases in DNA. Alkylation of guanine causes ionisation of the molecule and changes its base pairing specificities. Alkylated guanine pairs with thymine instead of cytosine.

Alkylation of purines leads to hydrolysis of the sugar base linkage so that the purine base is lost from the backbone of the DNA molecule producing a purinic gaps. When DNA replication takes place, almost any base may be inserted in the gap. Insertion of a wrong base in a gap produces transitions as well as transversions. There are some bifunctional alkylating agents which form cross links between guanines on the same or opposite strands of the double helix. This causes more frequent production of a purinic gaps.

Examples of alkylating agents are nitrosoguanidine, mustard gas and mustard compounds, ethyl methane sulphonate (EMS) and ethyl ethane sulphonate (EES), alkyl halides, sulphuric and phosphoric esters, ethylene imines and amides, and others. They are said to be electrophilic (electron deficient) reactants because they combine with nucleophiles which have electron rich centers. These compounds are also described as radiomimetic because their effects resemble those of ionising radiation.

The extent of a mutation can range from a change to a single base pair (point mutation) to the alteration of a large region of a chromosome (chromosome aberration). Mutations can occur anywhere in the total DNA of an organism. In humans, approximately 95% of DNA does not code for any gene products. As a result, many mutations have no effect on the phenotype, because they are located in regions of the genome that have no impact on cellular functions. By contrast, a mutation within an exon of a structural gene can alter the functioning of the gene product and cause a dramatic phenotypic change.

A point mutation is a change in a single base pair in DNA.


A change in a single nitrogenous base can change the entire structure of a protein

mRNA
Normal

Protein Replace G with A mRNA

Point mutation

Protein

Mutations have been broadly categorised as somatic and germ line mutations. When a mutation occurs in a somatic cell, it does not change the whole organism, but produces a phenotypic change in the organ to which the mutant cell belongs. The resulting individual is a mosaic for mutant and normal tissues. Germinal mutations take place in cells of the germ line and could be transmitted by gametes to the next generation.

Mutations which alter nucleotide sequences within a gene are of two types: base pair substitutions and frame shift mutations. In base pair substitution one base pair, for example AT may be replaced by another such as CG or GC. These are of two further types, namely transitions and transversions. Transitions are base changes in which a purine is substituted by another purine, or when a pyrimidine is replaced by a pyrimidine Transversions are alterations in bases in which a purine is substituted by a pyrimidine, that is, when an A = T pair is replaced either by T = A or C = G, and vice versa.

In frame shift mutations, (so called because they shift the normal reading frame of base triplets in mRNA) single base pairs are deleted from or added to DNA in interstitial position. The genetic code requires reading of consecutive base triplets from a fixed starting point. If a single nucleotide is inserted or deleted, it shifts the reading frame, and all the subsequent triplets are read off differently.

In general,DNA codon mutations are classified as: - silent - neutral - missense - nonsense

A silent mutation occurs when there is a change of a DNA codon, but the amino acid that is inserted into the protein is not changed In a number of these cases, the difference between the codons for a single amino acid lies in the third position, where U, G, C, or A can be present without altering the specificity of the codon. A silent mutation has no impact on the function of the protein.

A neutral mutation represents a nucleotide change at the DNA level that alters a codon so that another amino acid is incorporated into the protein with no apparent loss of function. This type of change is tolerated if the substitution occurs in a part of the protein that is not important for its function or if the alternative amino acid has physicochemical properties similar to the original one, as when valine substitutes for leucine.

A base pair substitution producing a codon that specifies another amino acid is a missense mutation. The severity of a missense mutation depends on the nature of the substituted amino acid and whether the original amino acid plays an essential role in the function of the protein. A neutral mutation is a missense mutation with no obvious consequences.

A nonsense mutation occurs when a nucleotide substitution changes a codon that specifies an amino acid into one that is a stop codon. The presence of a stop codon within an mRNA causes an incomplete (truncated) protein to be produced. If a nonsense mutation is near the N-terminus, it is unlikely that a functional protein fragment will be synthesized. However, if it is near the C-terminus, an incomplete polypeptide chain might be produced with some limited activity.

When a mutation changes the wild type normal genotype to a mutant type, as is more often the case, the event is called a forward mutation. This is in contrast to reverse mutations in which the mutant genotype changes to the original wild type. In a true reverse mutation, the original base pair sequence of the wild type may be restored. Thus if a GC pair of the wild type sequence is replaced by an AT pair to produce a forward mutation, a true reverse mutation could again substitute a GC pair in that position.

The purpose of a genetic diagnostic test is to identify a specific component (indicator, target) that signifies whether an individual has or will develop a particular disorder.

Genetic tests are used to: Establish the basis of an existing disorder (diagnostic testing) Determine the presence of genetic condition when there are no obvious symptoms (predictive testing) Identify heterozygotes (carrier testing) Assess a fetus for abnormalities (prenatal testing)

A genetic test is any diagnostic procedure that is used to determine an inherited disorder. The target may be a chromosome, protein, metabolite, or nucleic acid (RNA, DNA) sequence

Biochemical and immunological assays of the blood and urine detect amino acids, organic acids, metabolites, or proteins that are specifically associated with genetic disorders.

Currently, with a variety of formats, analytic procedures are able to detect more than 40 genetic diseases. The Guthrie test, a microbial assay that was developed by the US scientist Robert Guthrie (19101995), has been used for over 30 years for the diagnosis of phenylketonuria (PKU) in newborns. For this screening procedure, drops of blood, usually from a newborn infant, are spotted as circles on a paper card. The spots are air-dried, punched out in the form of disks, and placed on an agar surface covered with the bacterium Bacillus subtilis.

This bacterial strain requires phenylalanine for growth. The agar contains b-2-thienylalanine that inhibits the uptake of phenylalanine by the bacteria. If the blood on a disk contains a high level of phenylalanine, the phenylalanine diffuses into the agar and blocks the inhibitor. Under these conditions, a halo of bacterial cells forms around the periphery of the disk. A disk with a normal blood level of phenylalanine does not have a surrounding zone of bacterial growth because there is not enough phenylalanine to block the growth inhibitor.

Restriction endonuclease assays - loss or gain of restriction endonuclease site - PCR-based creation of restriction endonuclease site Oligonucleotide ligation assays Allele-specific amplification (ASA) - amplification refractory mutation system (ARMS) - mutagenically-separated PCR (MS-PCR) Allele-specific oligonucleotide (ASO) arrays - DNA chips - multiplex allele-specific diagnostic assay (MASDA) - Minisequencing - arrayed primer extension (APEX) DNA sequencing

The initial ability to manipulate Genetic material came in the early 1970s with the simultaneous development of three techniques.

The ability to transform E. coli The cutting and joining of DNA molecules The ability to monitor these reactions.

The DNA needed to be detectable once transformed into the host organism The DNA needed to be replicated with the host cell once transformed Methods were required for detecting the presence and size of DNA molecules.

DNA (containing genes of interest to transformation), which was to be used to transform a organism was first integrated into an artificial replicon. These include Plasmid and Phage Vectors in bacterial, and artificial chromosomes in other eukaryotes.

In order for target DNA molecule to be inserted into a plasmid:


Both the vector and target DNA must be, purified, cut and rejoined. These reactions must be monitored and this is achieved by Gel electrophoresis.

DNA molecules in vitro, have a net negative charge DNA molecules in an electric field move towards the negative terminal. DNA molecules of differing size move through an agarose gel at different rates (larger = slower). Thus agarose gel electrophoresis can separate DNA molecules based on their size

A reaction which exploits the properties of the enzyme Thermophilic DNA polymerase (Taq), to amplify a known region of DNA of known sequence or flanked by known sequence. PCR forms a cornerstone of modern molecular biology, allowing for the rapid amplification of DNA sequences. Short sections of DNA of complementary known as Primers, bind to the target DNA sequence and facilitate the extension of the Target DNA by Taq.

The transformation of E. coli can be achieved via a number of different methods. The two main methods involve: Electroporation: subjecting the E. coli cells to an electric shock in the presence the target DNA Heat Shock: Subjecting the E. coli cells to a rapid change in temperature (0-5 to 37-45oC) in the presence of CaCl2 and the Target DNA.

Transformation of E. coli provides a useful means for studying genetics as well as the production of useful proteins and metabolites. E. coli is also invaluable as mean of amplifying (or cloning) a DNA molecules for transformation of another organism.

The Insertion of plasmid into bacteria is useful for a number of reasons.


Amplification of foreign DNA for further experiments. The production of recombinant protein in bacterial cells (discussed in subsequent tutorials) Study of model biochemical systems in bacteria.

Plasmids are replicons stably inherited in an extrachromosomal state, they are found in many species of bacteria Usually exist in a bacterial cell as a Closed Covalent Circle (CCC DNA), but linear plasmids do exist. The CCC DNA is often supercoiled and this effects its movement through an agarose gel and density when processed with ethidium bromide.

Some phenotypic traits exhbited by plasmid-carried genes. Antibiotic resistance Antibiotic production Degradation of aromatic compounds Haemolysin production Enterotoxin production Heavy-metal resistance Bactericin production Induction of plant tumors Hyrdogen sulphide production

Example of a Plasmid

The States of Plasmid DNA

Most plasmids do not contain all of the proteins (Enzymes required for their replication, the remainder are provided by the bacteria. This limits their host range to Bacterial species whos enzymes can recognize the plasmids origin of replication (ori). The number of copies of a plasmid which are present in a bacterial cell is also dependant on its origin of replication. I.e. how often the Plasmid is allowed to replicate.

Low Molecular Weight


Easier to handle, Large DNA molecules shear more easily Less energy required for bacterial cell to replicate plasmid Generally allows higher copy number per cell

Ability to confer selectable trait on host cells


Allows successful transformation to be selected for Blue/ White Selection Antibiotic Resistance Metabolic Advantage

Single area of the Plasmid containing many unique restriction sites.


Allows for the flexible insertion of target DNA into Plasmid

It is necessary to determine if a bacterial cell has been successfully transformed with a plasmid. This is usually achieved via an antibiotic resistance gene being included on the plasmid Many plasmids naturally contain these genes and they generally have been extracted from these plasmids and engineered into commercially available plasmids Bacteria which have been transformed with a plasmid can be transferred to agar which contains an antibiotic, which the plasmid confers resistance. Only bacteria which have been successfully transformed will grow.

As well as detecting the presence of a plasmid in a bacterial line, it is often also useful to determine if the the target DNA has been inserted into the plasmid. This can be achieved by placing a gene which contains a detectable trait in the insert region of the plasmid. Thus in plasmid molecules where this disruption occurs, there will be a noticeable change in phenotype. An example of this exploits an enzyme responsible for the cleavage of lactose in E. coli (Contained on in the LacZ operon). This enzyme will also cleave an artificial substrate (X-gal), which will lead to the formation of a blue colour. This phenotype can be screened for by including X-gal in the E. coli grown media directly after transformation.

Native plasmids, i.e. those originally isolated from bacteria have limited usefulness for cloning. This is due to size, low copy number or the presence of undesirable attributes (Genes encoding conjugation pili.) Also the presence of multiple common restriction enzyme sites, or sites in regions of essential sequence (I.e. The origin of replication.) Because of this considerable effort has gone into engineering plasmids suitable for cloning. This has been an ongoing process and currently there are many commercially custom built plasmids for cloning.

Lokasi : Lab Mikrobiologi Kegiatan : - PCR - Elektroporesis - Interpretasi hasil Tugas : Membuat laporan tentang kegiatan tersebut