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Genetics

Instructions:
Detailed study notes, guide and class notes on genetic engineering.

The application of polymerase chain reaction( PCR) and it's applications.
Content:

ABT 4301: GENETIC ENGINEERING 1

ABT 4308 Genetic Engineering 1

Introduction

The gene

A gene is defined as a heritable unit of phenotypic variation. From a molecular standpoint, a gene is the linear DNA sequence required to produce a functional RNA molecule, or a single transcriptional unit.

Genes can be assigned to one of two broad functional categories: structural genes and regulatory genes. It is the function of the end product of a gene that distinguishes structural and regulatory genes.

Structural genes code for polypeptides or RNAs needed for the normal metabolic activities of the cell, e.g. enzymes, structural proteins, transporters, and receptors, among others.

Regulatory genes code for proteins whose function is to control the expression of structural genes. With regard to molecular composition both classes of genes are similar.

A gene usually occupies a defined location on a chromosome, of which there are 46 in every human cell and which contain the entire human genome. The exact chromosomal gene location is defined by specific sequences for the start and termination of its transcription.

Each gene has a specific effect and function in the organism’s morphology or physiology, can be mutated (i.e. changed), and can recombine with other genes.

It is a store of information (in the form of nucleotide base sequence) and the complete set of genes of an organism, its genetic constitution, is called the genotype.

The physical manifestation, or expression, of the genotype is the phenotype (i.e. the organism’s morphology and physiology). If a particular characteristic, such as brown eye colour, is part of an organism’s phenotype, one can conclude that the individual carries the gene(s) for that characteristic. Different varieties of the same gene, resulting in different phenotypic characteristics, are called alleles.

Genes may be located on either strand of the double-stranded DNA. But, regardless of which strand contains a particular gene, all genes are read in a 5’ to 3’ direction, and the strand containing the particular gene is referred to as the sense or coding strand.

Every cell of a human body, except germ line cells, contains 46 chromosomes. From each parent, we inherit 23 chromosomes, representing the complete genome. Thus, each body cell is diploid, i.e. contains two copies of the human genome and likewise two copies (alleles) of each gene.

In comparison, the genome of E. coli, a widely used model bacterium, consists of one chromosome of 4.6 Mb in size, encoding approximately 4 400 genes in total. The genome of Arabidopsis thaliana, probably the most important model plant, consists of five chromosomes, of 157 Mb in size and encodes approximately 27 000 genes.

The arrangement and layout of genes

Along the length of each DNA molecule/chromosome one can find thousands of genes, with more or less random spacing. In bacteria, one can frequently find clusters of genes that are related, in the sense that the proteins encoded by these genes are required in the same metabolic pathway. Therefore, as the cell needs all the gene products more or less simultaneously in order to keep that pathway running, it is appropriate for the cell to arrange these genes in clusters and employ a mechanism to express them together. These clusters of genes are known as operons; the most studied operon is the lactose operon in E. coli. This operon contains three genes which are adjacent on the DNA and are required for the utilization of lactose as a metabolic energy source in the cell.

Many genes in eukaryotes have a distinctive structural feature: the nucleotide sequence contains one or more intervening segments of DNA that do not code for the amino acid sequence of the protein. Such non-translated DNA segments in genes are called introns. The pieces that constitute mature mRNA, and therefore ultimately for protein, are referred to as exons.

In addition to introns and exons, the structural features of the eukaryotic gene include regulatory elements, a promoter region, a transcription start site and a transcription termination site.

The promoter region is the sequence of the gene where the transcription machinery (the assembly of proteins required for transcription) binds to the DNA in order to start transcription to RNA. The start site indicates to the transcription machinery where to start and the termination site indicates where to stop transcription of the gene.

Gene expression

Genes exert their function through a process called gene expression, a process by which heritable information from a gene, encoded on DNA, is transformed into a functional gene product, such as protein or RNA (some genes code for functional RNA molecules, such as tRNA and rRNA).

Genes are expressed by being first transcribed into RNA, and may then subsequently be translated into protein. A cell employs many different mechanisms to regulate gene expression.

A general structural arrangement of the different components making up a eukaryotic gene

Upstream regulatory elements (enhancers) and the promoter are required for regulation and initiation of transcription.

Exons, which constitute the actual protein-coding regions, and interspersed introns are indicated.

The 5’ and 3’ untranslated regions (UTRs) are mRNA sequences that do not encode protein, but are required for a correct translation process. Transcription start and termination sites are also indicated.



Expression can be regulated at many different levels, from DNA transcription, pre-mRNA processing, mRNA stability and efficiency of translation up to protein modification and stability. Thus, a cell can precisely influence the expression level of every gene, and studying and predicting gene expression levels is a difficult task. Nevertheless, this is especially important for biotechnological applications, since it is desirable to precisely define the expression levels of introduced genes in transgenic organisms.

Transcription and translation

The first step in gene expression is transcription, namely the production of a single stranded RNA molecule known as mRNA in the case of protein-coding genes. The nucleotide sequence of the mRNA is complementary to the DNA from which it was transcribed.

The DNA strand whose sequence matches that of the RNA is known as the coding strand and the complementary strand on which the RNA was synthesized is the template strand.

Transcription is performed by an enzyme called RNA polymerase, which reads the template strand in 3’ to 5’ direction and synthesizes the RNA from 5’ to 3’ direction. To initiate transcription, the polymerase first recognizes and binds a promoter region of the gene. Thus a major regulatory mechanism of gene expression is the blocking or sequestering of the promoter region.

Translation

Is the process by which a mature mRNA molecule is used as a template for synthesizing a protein.

Translation is carried out by the ribosome, a large macromolecular complex of several rRNA and protein molecules. Ribosomes are responsible for decoding the genetic code on the mRNA and translating it into the amino acid sequence of proteins. The genetic code on the mRNA is read three nucleotides at a time, in units called codons, via interactions of the mRNA with specialized RNA molecules called transfer RNA (tRNA).

Each tRNA has three unpaired bases, known as the anticodon, that are complementary to the codon it reads. The tRNA is also covalently attached to the amino acid specified by its anticodon. When the tRNA binds to its complementary codon in an mRNA strand, the ribosome ligates its amino acid cargo to the growing polypeptide chain. When the synthesis of the protein is finished, as encoded by a stop-codon on the mRNA, it is released from the ribosome.

During and after its synthesis, the new protein must fold to its active three-dimensional structure before it can carry out its cellular function. A single mRNA molecule can be translated several times and thus produce many identical proteins, depending on its half-life in the cell, i.e. the average time it remains within the cell before it is degraded.

Regulation of gene transcription

Promoter A DNA sequence associated with a gene that is responsible for recruiting the enzyme machinery required for the expression of that gene.

Enhancers DNA sequences that influence the expression of a gene, often over long distances of DNA sequence.

Operators are nucleotide sequences that are positioned between the promoter and the structural gene. They constitute the region of DNA to which repressor proteins bind and thereby prevent transcription.

Repressor proteins have a very high affinity for operator sequences. Repression of transcription is accomplished by the repressor protein attaching to the operator sequence downstream of the promoter sequence (the point of attachment of the RNA polymerase).

Attenuators The attenuator sequences are found in bacterial gene clusters that code for enzymes involved in amino acid biosynthesis. Attenuators are located within so-called leader sequences, a unit of about 162 bp situated between the promoter-operator region and the start site of the first structural gene of the cluster. Attenuation decreases the level of transcription approximately 10-fold. As the concentration of an amino acid in the cell rises and falls, attenuation adjusts the level of transcription to accommodate the changing levels of the amino acid. High concentrations of the amino acid result in low levels of transcription of the structural genes, and low concentrations of the amino acid result in high levels of transcription. Thus the biosynthesis of an amino acid can be linked to the actual concentration of that amino acid within the cell. Attenuation proceeds independently of repression, the two phenomena are not dependent on each other. Attenuation results in the premature termination of transcription of the structural genes

Genetic Engineering

Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a new genotype.

Various kinds of genetic modification are possible:

inserting a foreign gene from one species into another

altering an existing gene so that its product is changed

changing gene expression so that it is translated more often or not at all.

Principles of genetic engineering

Genetic engineering, also known as recombinant DNA (rDNA) technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.

Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all.

 Basic steps in genetic engineering

Isolate the gene

Insert it in a host using a vector - molecule of DNA which is used to carry a foreign gene into a host cell

Produce as many copies of the host as possible

Separate and purify the product of the gene

Possible applications of rDNA technology

Areas:

Agriculture - Production of GM crops that are resistant to insects, drought, herbicides, salts etc

Cloning of gene and mass production of recombinant proteins

Gene therapy

Bioremediation through use of genetically engineered organisms.

Mass production of useful protein such as insulin for treatment of diabetes and other protein for industrial use e.t.c

DNA isolation methods

Many different methods and technologies are available for the isolation of genomic DNA. In general, all methods involve disruption and lysis of the starting material followed by the removal of proteins and other contaminants and finally recovery of the DNA.

Removal of proteins is typically achieved by digestion with proteinase K, followed by salting-out, organic extraction, or binding of the DNA to a solid-phase support (either anion-exchange or silica technology).

DNA is usually recovered by precipitation using ethanol or isopropanol. The choice of a method depends on many factors:

the required quantity and molecular weight of the DNA, the purity required for downstream applications, and the time and expense.

Several of the most commonly used methods are detailed below, although many different methods and variations on these methods exist.

Home-made methods often work well for researchers who have developed and regularly use them. However, they usually lack standardization and therefore yields and quality are not always reproducible.

Reproducibility is also affected when the method is used by different researchers, or with different sample types. The separation of DNA from cellular components can be divided into four stages:

Disruption

Lysis

Removal of proteins and contaminants

Recovery of DNA

NB: In some methods, stages 1 and 2 are combined.

Conventional Methods

(1) Guanidinium Thiocyanate-Phenol-Chloroform Extraction.

Proteins, lipids, carbohydrates, and cell debris are removed through extraction of the aqueous phase with the organic mixture of phenol and chloroform. A biphasic emulsion forms when phenol and chloroform are added. The hydrophobic layer of the emulsion will then be settled on the bottom and the hydrophilic layer on top by centrifugation. The upper phase which contained DNA is collected and DNA can be precipitated from the supernatant by adding ethanol or isopropanol in 2:1 or 1:1 ratios and high concentration of salt.

(2) Alkaline Extraction Method

Alkaline lysis has been used to isolate plasmid DNA and E. coli. It works well with all strains of E. coli and with bacterial cultures ranging in size from 1 mL to more than 500 mL in the presence of Sodium Dodecyl Sulfate (SDS).

The principle of the method is based on selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double stranded. Bacterial proteins, broken cell walls, and denatured chromosomal DNA enmeshed into large complexes that are coated with dodecyl sulfate. Plasmid DNA can be recovered from the supernatant after the denatured material has been removed by centrifugation.

(3) CTAB Extraction Method

For plant extraction, the initial step that needs to be done is to grind the sample after freezing it with liquid nitrogen. The purpose of doing this step is to break down cell wall material of sample and allow access to nucleic acid while harmful cellular enzymes and chemicals remain inactivated.

This method also uses organic solvents and alcohol precipitation in later steps. Insoluble particles are removed through centrifugation to purify nucleic acid. Soluble proteins and other material are separated through mixing with chloroform and centrifugation.

(5) Ethidium Bromide (EtBr)-Cesium Chloride (CsCl) Gradient Centrifugation.

Genomic DNA can be purified by centrifugation through a cesium chloride (CsCl) density gradient. Cells are lysed using a detergent, and the lysate is alcohol precipitated. Resuspended DNA is mixed with CsCl and ethidium bromide and centrifuged for several hours. Intercalation of EtBr alters the swimming density of the molecule in high molar CsCl. Covalently closed circular molecules will accumulate at lower densities in the CsCl gradient because they incorporate less EtBr per base pair compared to linear molecules.

NB: Below is a list of some of the chemical substances and their roles in DNA isolation

Isotonic buffer: The isotonic property of the solution prevents the cells from bursting due to osmosis, and the buffer prevents the cells from being damaged by pH changes. A common isotonic buffer used in DNA isolation is buffered sucrose.

EDTA: This is a common food additive and laboratory reagent. It binds to and removes positive ions such as magnesium (Mg2+) and calcium (Ca2+) ions. These ions strengthen membranes by neutralizing the negative charges on the phospholipids, so removing these ions weakens the cellular and nuclear membranes.

SDS detergent: Recall from your lab on lipids that detergents and soaps are molecules that break apart globules of lipids. Detergent molecules are attracted to both lipids and water, by binding to both lipid and water the detergent is able to dissolve the membranes of the cell. In other words, detergents lyse (break open) cells membranes and nuclear membranes. The most common detergent used in biology laboratories (including today’s DNA isolation lab) is called sodium dodecyl sulfate (SDS).

Sodium Chloride (NaCl, table salt): The detergents and the EDTA dissolve the cell’s membranes, which releases the genomic DNA. But the released DNA is not pure. The cell’s proteins are still present. To make matters worse, the proteins tend to form ionic bonds to the DNA, so they stick to the DNA and contaminate its purity. As a first step in separating the proteins from the DNA, a large amount of NaCl salt (which is Na+ and Cl- ions) is added. The Na+ ions neutralize the negative charge onthe DNA, whereas the Cl- ions neutralize the positive charges on the proteins. With their charges neutralized, the proteins and DNA no longer form strong ionic bonds to one another and therefore can be easily separated.

Alcohol: After the NaCl has separated the proteins from the DNA, the final step in DNA isolation is usually to add ice cold alcohol to the mixture. The alcohol forms a separate layer on top of the cell homogenate. The DNA is not soluble in the alcohol layer, so it will form a precipitate (a mucus-like solid in the alcohol layer). The proteins, on the other hand, do not precipitate when the alcohol is added (they stay dissolved in the homogenate). The precipitated DNA can therefore be separated from the proteins by spooling it onto a rod, then the rod is used to transfer the spooled DNA into sterile water. The DNA has now been successfully isolated from the cell.

NB: Although not “chemicals”, there are two more features of most DNA isolations that help protect the DNA.

The first is coldness: The DNA isolation procedure is usually done on ice to slow the action of cellular enzymes that that might degrade the DNA.

The second is gentleness: DNA strands are extremely long and fragile. Therefore all steps should be done slowly and gently to avoid shearing the DNA strands.

2. Solid-phase Nucleic Acid Extraction

cell lysis,

nucleic acids adsorption,

washing, and

elution

Introduction to Gel Electrophoresis

Agarose gel electrophoresis:

A method used in biochemistry and molecular biology to separate DNA or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electrotric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones.

Equipments and Supplies

•Buffer solution, usually TBE buffer or TAE 1.0x, pH 8.0

•Agarose

•An ultraviolet-fluorescent dye, ethidium bromide, (5.25 mg/ml in H2O). Alternative dyes may be used, such as SYBR Green.

•Nitrile rubber gloves. Latex gloves do not protect well from ethidium bromide

•A color marker dye containing a low molecular weight dye such as "bromophenol blue“

•A gel rack

•A "comb“

•Power Supply

•UV lamp or UV lightbox or other method to visualize DNA in the gel

Application of gell electrophoresis

Estimation of the size of DNA molecules

Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting

Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern analysis

Factors affecting gel electrophoresis

DNA or RNA Molecular Weight

Voltage

Agarose

Buffer

Visualisation

DNA or RNA Molecular Weight

The length of the DNA molecule is the most important factor, smaller molecules travel farther and vice versa.

Voltage

The higher the voltage, the faster the DNA moves. But voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm)

Agarose

Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules.

The distance between DNA bands of a given length is determined by the percent agarose in the gel. In general lower concentrations of agarose are better for larger molecules because they result in greater separation between bands that are close in size. The disadvantage of higher concentrations is the long run times (sometimes days). Instead high percentage agarose gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.

Concentration of most agarose gels:

1% gels are common for many applications.

0.7%: good separation or resolution of large 5–10kb DNA fragments

2% good resolution for small 0.2–1kb fragments.

Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case.

Buffer

The most common buffers for agarose gel are:

TAE: tris acetate EDTA

TBE:: Tris/Borate/EDTA

SB: Sodium borate

TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product.

Visualization

The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than~20ng DNA becomes distinctly visible. EtBr is a known carcinogen, however, safer alternatives are available.



A color marker dye containing a low molecular weight dye such as "bromophenol blue" (to enable tracking the progress of the electrophoresis) and glycerol (to make the DNA solution denser so it will sink into the wells of the gel).

The advantages are that the gel is easily poured, does not denature the samples. The samples can also be recovered.

The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms. After the experiment is finished, the resulting gel can be stored in a plastic bag in refrigerater.

Detecting DNA molecules

Once you have your DNA separated by size, you need to be able to visualize the DNA on the gel somehow. Original techniques include radioactive label, silver staining. Current techniques include, fluorescent dyes etc.



Radioisotopes

• Incorporate P32 into one of the dNTPs. The DNA strand that is made from PCR reaction will contain radiolabel. Either blot the gel or dry the gel itself. Expose the gel or the blot to photographic film –keep it in the freezer, overnight. Radiation will expose photographic film leaving dark bands on the film wherever there are DNA bands in the gel

Can detect the bands – very sensitive

Disadvantages:

Radioactivity is dangerous

Expensive and wasteful

Takes long time

Only have one color band-therefore need to have everything separated completely based on size first

Silver Staining

• Run your PCR products through a gel to separate based on size. Then expose gel to silver solution-Silver will bind to DNA within the gel matrix. Photo develop the gel until silver becomes dark and visible with the naked eye. Take a picture of the gel for permanent record of the DNA bands.

Advantages to Silver Staining

Reagents are less dangerous than radioactivity

Less expensive than radioactivity

In fact Silver Staining is one of the

cheapest ways to detect DNA bands

Staining is quick

Within 30 minutes

Sensitivity is better than ethidium bromide

Not as good as radioactivity

Disadvantages to Silver Staining

Major disadvantage is that there is only one color

Therefore need to have everything separated completely based on size first

Also, silver binds to both strands of DNA

Heterozygotes are difficult to visualize correctly on the gel

Fluorescence

• Currently – everyone uses fluorescence to visualize DNA

Main reasons:

1. More than one colored dye can be used-allowing multiplexing

2. Detection can be automated

• The laser that excites the dye can also read the band of the gel. A specific color of fluorescent dye is attached to one of the PCR primers. This way the dye is incorporated into the PCR product

• PCR products are separated on a gel or capillary based on size

• Laser excites the dye within each product– Also measures the light being emitted by each dye

Other methods of DNA detection

Measuring the intensity of absorbance of the DNA solution at wavelengths  HYPERLINK "http://en.wikipedia.org/wiki/Quantification_of_nucleic_acids" \o "Quantification of nucleic acids" 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs  HYPERLINK "http://en.wikipedia.org/wiki/UV" \o "UV" UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has a ratio of 1.8 at 260/280 and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

DNA can be quantified by cutting the DNA with a  HYPERLINK "http://en.wikipedia.org/wiki/Restriction_enzyme" \o "Restriction enzyme" restriction enzyme, running it on an agarose  HYPERLINK "http://en.wikipedia.org/wiki/Gel_electrophoresis" \o "Gel electrophoresis" gel, staining with  HYPERLINK "http://en.wikipedia.org/wiki/Ethidium_bromide" \o "Ethidium bromide" ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.

NB: Using the  HYPERLINK "http://en.wikipedia.org/wiki/Southern_blot" \o "Southern blot" Southern blot technique, this quantified DNA can be isolated and examined further using  HYPERLINK "http://en.wikipedia.org/wiki/PCR" \o "PCR" PCR and  HYPERLINK "http://en.wikipedia.org/wiki/RFLP" \o "RFLP" Restriction Fragment Length Polymorphysm (RFLP) analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which  HYPERLINK "http://en.wikipedia.org/wiki/Forensic" \o "Forensic" forensic scientists use for comparison, identification, and analysis.

DNA concentration

Quantification of nucleic acids is commonly used in  HYPERLINK "http://en.wikipedia.org/wiki/Molecular_biology" \o "Molecular biology" molecular biology to determine the concentrations of  HYPERLINK "http://en.wikipedia.org/wiki/DNA" \o "DNA" DNA or  HYPERLINK "http://en.wikipedia.org/wiki/RNA" \o "RNA" RNA present in a mixture, as subsequent reactions or protocols using a  HYPERLINK "http://en.wikipedia.org/wiki/Nucleic_acid" \o "Nucleic acid" nucleic acid sample often require particular amounts for optimum performance.

There exist several methods to establish the concentration of a solution of nucleic acids, including spectrophotometric quantification and UV fluorescence in presence of a DNA dye.

DNA concentration can be determined by measuring the intensity of absorbance of the solution at the 600 nm with a  HYPERLINK "http://en.wikipedia.org/wiki/Spectrophotometer" \o "Spectrophotometer" spectrophotometer and comparing to a  HYPERLINK "http://en.wikipedia.org/wiki/Standard_curve" \o "Standard curve" standard curve of known DNA concentrations.

Measuring the intensity of absorbance of the DNA solution at wavelengths  HYPERLINK "http://en.wikipedia.org/wiki/Quantification_of_nucleic_acids" \o "Quantification of nucleic acids" 260 nm and 280 nm is used as a measure of DNA purity. DNA absorbs  HYPERLINK "http://en.wikipedia.org/wiki/UV" \o "UV" UV light at 260 and 280 nanometres, and aromatic proteins absorb UV light at 280 nm; a pure sample of DNA has the 260/280 ratio at 1.8 or (between1.6-2.0) and is relatively free from protein contamination. A DNA preparation that is contaminated with protein will have a 260/280 ratio lower than 1.8.

DNA can be quantified by cutting the DNA with a  HYPERLINK "http://en.wikipedia.org/wiki/Restriction_enzyme" \o "Restriction enzyme" restriction enzyme, running it on an agarose  HYPERLINK "http://en.wikipedia.org/wiki/Gel_electrophoresis" \o "Gel electrophoresis" gel, staining with  HYPERLINK "http://en.wikipedia.org/wiki/Ethidium_bromide" \o "Ethidium bromide" ethidium bromide or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration. Using the  HYPERLINK "http://en.wikipedia.org/wiki/Southern_blot" \o "Southern blot" Southern blot technique this quantified DNA can be isolated and examined further using  HYPERLINK "http://en.wikipedia.org/wiki/PCR" \o "PCR" PCR and  HYPERLINK "http://en.wikipedia.org/wiki/RFLP" \o "RFLP" RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. These are the techniques which  HYPERLINK "http://en.wikipedia.org/wiki/Forensic" \o "Forensic" forensic scientists use for comparison, identification, and analysis.

Sample purity

Some contaminants (notably  HYPERLINK "http://en.wikipedia.org/wiki/Phenol" \o "Phenol" phenol) can significantly contribute to an error in concentration estimation as they also absorb strongly at 260nm.

Protein contamination and the 260:280 ratio

The ratio of absorptions at 260nm vs 280nm is commonly used to assess the purity of DNA with respect to  HYPERLINK "http://en.wikipedia.org/wiki/Protein" \o "Protein" protein contamination, since protein (in particular, the aromatic amino acids) tends to absorb at 280nm.

Contamination by  HYPERLINK "http://en.wikipedia.org/wiki/Phenol" \o "Phenol" phenol, which is commonly used in nucleic acid purification, can significantly throw off quantification estimates. Phenol absorbs with a peak at 270nm and a 260:280 ratio of 2. Nucleic acid preparations uncontaminated by phenol should have a 260:270 ratio of around 1.2. Contamination by phenol can significantly contribute to overestimation of DNA concentration.

Absorption at 230nm can be caused by contamination by  HYPERLINK "http://en.wikipedia.org/wiki/Phenolate" \o "Phenolate" phenolate ion,  HYPERLINK "http://en.wikipedia.org/wiki/Thiocyanates" \o "Thiocyanates" thiocyanates, and other organic compounds. For a pure nucleic acid sample, the 260:230 ratio should be around 2.

Absorption at 330nm and higher indicates particulates contaminating the solution, causing scattering of light in the visible range. The value in a pure nucleic acid sample should be zero.

Negative values could result if an incorrect solution was used as blank. Alternatively, these values could arise due to fluorescence of a dye in the solution.

The Polymerase Chain Reaction (PCR)

Introduction

NB: Nucleotides are the building stones of DNA.

There are 4 different nucleotides :

dATP : deoxyadenosine triphosphate

dGTP : deoxyguanosine triphosphate

dTTP : deoxythymidine triphosphate

dCTP : deoxycytidine triphosphate

For convenience, these 4 nucleotides are called dNTP's (deoxynucleoside triphosphates). A nucleotide is made of three major parts : a nitrogen base, a sugar molecule and a triphosphate. Only the nitrogen base is different in the 4 nucleotides.

DNA is formed by coupling the nucleotides between the phosphate group from a nucleotide (which is positioned on the 5th C-atom of the sugar molecule) with the hydroxyl on the 3rd C-atom on the sugar molecule of the previous nucleotide. To accomplish this, a diphosphate molecule is split off (and releases energy). This means that new nucleotides are always added on the 3' side of the chain.

The PCR Reaction Components

Despite the numerous variations on the basic theme of PCR, the reaction itself is composed of only a few components. These are as follows:

• Sterile deionised water

• 10X PCR buffer

• dNTP mix (range from 100 – 500 (M)

• Primer (range from 0.1 – 0.5 (M)

• Taq DNA polymerase (range of 1-2.5 Units/ 100(l)

• MgCl2 (range from 1.5 – 2.0 mM)

• Template DNA (final concentration: 1-10 (g/ml)

• Bovine serum albumin (BSA)

MgCl2 – Acts as an activator for the enzyme Taq polymerase i.e it enhances its activity.

Taq polymerase – The enzyme that synthesizes new DNA strands complementary to the DNA template by adding bases complementary to the template.

Bovine serum albumin – type of protein that counteracts (removes) protein contaminants in a PCR reaction mixture.

Primers – Short nucleotide sequences that are complementary to the 5’ end of one template and the 3’ end of the other template, which flank the target gene.

Many PCR machines are now available in 48-, 96- or 384-well formats. This, combined with the use of multichannel pipettors, can greatly increase the number of reactions that can be done simultaneously. If several reactions need to be simultaneously prepared, a master mix should be used as follows:

Sterile distilled water, buffer, dNTPs, primers, MgCl2 and Taq DNA polymerase in a single tube. This will then be aliquoted into individual tubes.

PCR Considerations:

Template DNA

Nearly any standard method is suitable for template DNA purification.

An adequate amount of template DNA is between 0.1 and 1 µg for genomic DNA for a

total reaction mixture of 100 µl. Larger template DNA amounts usually increase the yield

of non-specific PCR products.

Primers

PCR primers should be 10-24 nucleotides in length.

The GC content should be 40%-60%.

The primer should not be self-complementary or complementary to any other primer in the reaction mixture, to prevent primer-dimer and hairpin formation.

Melting temperatures of primer pairs should not differ by more than 5°C, so that the GC content and length must be chosen accordingly.

The melting and annealing temperatures of a primer are estimated as follows: if the primer is shorter than 25 nucleotides, the approximate melting temperature is calculated with the formula: Tm = 4(G + C) + 2 (A + T).

The annealing temperature should be about 5°C lower than the melting temperature.

MgCl2 concentration

Because Mg2+ ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg2+ ions result in a low yield of PCR product, and too many will increase the yield of non-specific products.

The recommended range of MgCl2 concentration is 1 to 3 mM, under the standard reaction conditions specified.

Taq DNA polymerase

Higher Taq DNA polymerase concentrations than needed may cause synthesis of non-specific products.

dNTPs

The concentration of each dNTP (dATP, dCTP, dGTP, dTTP) in the reaction mixture is usually 200 µM. These concentrations must be checked as being equal, because inaccuracies will increase the degree of mis-incorporation.

Lab equipment needed:

- Thermocycler (PCR machine)

- Thermocycler-specific tubes (individual or strips) or plate

- Micropipette with sterile tips

- a multi-channel pipette is also useful for some parts of the protocol if you are using a plate or tubes that are connected

- Electrophoresis units

- Power supply units

- Photographic equipment

Details of the PCR components

Water

PCR Reaction Buffer

so-called thermophile boundary which is set at 50-60oC. Williams defined several

terms that describe the relationship between temperature and growth rate for thermophilic bacteria.

thermophilic host bacteria so both native Taq, isolated from Thermus aquaticus, and

cloned Taq, isolated from expression systems in other bacteria, are commercially available. In addition, a number of other thermal-stable DNA polymerases, isolated from other thermophilic species, have become available. Among these are enzymes

from Pyrococcus furiosus (Pfu polymerase), Thermus thermophilus (Tth polymerase), Thermus flavus (Tfl polymerase), Thermococcus litoralis (Tli polymerase aka Vent polymerase), and Pyrococcus species GB-D (Deep Vent polymerase). Each of these, and other, polymerases has a specific set of attributes that can be selected depending upon the application. In general, there are three aspects of a DNA polymerase that should be considered. These are;

default processivity for Taq polymerase of 1000 nt per minute bearing in mind that setting your extension times for this assumed value is more than adequate. Many of the other polymerases listed above are slower than Taq. For example, Tth polymerase processivity is on the order of 25 nt per second.

Fidelity

polymerase also demonstrates fidelity in this range

Persistence

Refers to the stability of the enzyme at high temperature, is intimately linked to the other two polymerase attributes. Stability can be measured in terms of how long the enzyme retains at least one-half of its activity during sustained exposure to high temperature. Taq polymerase has a half-life of about an hour and a half at a sustained 95oC. Other enzymes have much longer half-lives. Tli polymerase has a half-life of over six hours and Deep Vent polymerase has shown a half-life of nearly a full day when exposed to a constant 95oC.

Principle of the PCR

The cycling reactions:

There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

i.  HYPERLINK "http://en.wikipedia.org/wiki/Denaturation_(biochemistry)" \l "Nucleic_acid_denaturation" \o "Denaturation (biochemistry)" Denaturation step: - This step is the first regular cycling event and consists of heating the reaction to 94 – 98 °C for 20–30 seconds. It causes  HYPERLINK "http://en.wikipedia.org/wiki/DNA_melting" \o "DNA melting" DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.

ii.  HYPERLINK "http://en.wikipedia.org/wiki/Annealing_(biology)" \o "Annealing (biology)" Annealing step: - Depends on the melting temperature of the primers. The reaction temperature is lowered to 45 – 65 °C for about 30–40 seconds allowing annealing of the primers to the single-stranded DNA template.

iii. Extension/elongation step: - The temperature at this step depends on the DNA polymerase used;  HYPERLINK "http://en.wikipedia.org/wiki/Taq_polymerase" \o "Taq polymerase" Taq polymerase has its optimum  HYPERLINK "http://en.wikipedia.org/wiki/Enzyme" \o "Enzyme" activity temperature at 75–80 °C, and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction.

Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on.

NB:

In the initial denaturation step, complete denaturation of the DNA template at the start of the PCR reaction is essential. Incomplete denaturation of DNA will result in the inefficient use of the template in the first amplification cycle and, consequently, poor yield of PCR product. The annealing temperature may be estimated as 5°C lower than the melting temperature of the primer-template DNA duplex.

If non-specific PCR products are obtained in addition to the expected product, the annealing temperature can be optimized by increasing it stepwise by 1-2°C.

Usually, the extension step is performed at 72°C and a 1-min extension is sufficient to synthesise PCR fragments as long as 2 kb (1kb = 1 kilobase = 1000 bp).

When larger DNA fragments are amplified, time is usually extended by 1 min per 1000 bp. The number of PCR cycles will basically depend on the expected yield of the PCR product. After the last cycle, samples are usually incubated at 72°C for 5 min to fill in the protruding ends of newly synthesized PCR products.

In a PCR reaction the amount of template DNA does not change while the number of anchored products increases arithmetically each cycle beginning with cycle 1. Beginning with cycle 2, when the first defined amplicons are formed, the number of defined amplicons increases at a geometric rate. This, then, is the explosive chain reaction from which PCR derives its name. At the end of 35 cycles there are more than 34 billion copies of the amplicon for every copy of the original template sequence in the reaction! Thus, if there are 10,000 copies of the target sequence, there are more than 340 trillion copies of the amplicon.

DNA extraction and PCR reaction mixing and processing should be performed in separate areas.

Use of sole-purpose vessels and positive displacement pipettes or tips for DNA sample and reaction mixture preparation is strongly recommended.

All solutions, except dNTPs, primers and Taq DNA polymerase, should be autoclaved. Where possible, solutions should be aliquoted in small quantities and stored in designated PCR areas.

Some DNA however cannot be stably maintained in E. coli, for example very large DNA fragment, and other organisms such as yeast may be used. Cloning vectors in yeast include yeast artificial chromosomes (YACs).

Features of cloning vector

Most commonly used cloning vectors 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 .

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) which contains many unique restriction sites.

NB:- The restriction sites in the MCS are first cleaved by restriction enzymes, and a PCR-amplified target gene, also digested with the same enzymes, is then ligated into the vectors using DNA ligase.

Selectable marker

A selectable marker is carried by the vector to allow the selection of positively transformed cells.

Antibiotic resistance is often used as a marker; an example is the beta-lactamase gene which confers resistance to the penicillin group of beta-lactam antibiotics like ampicillin.

Ampicillin inhibits synthesis of bacterial cell wall. amp resistance depends upon production of an enzyme that catalyzes beta-lactam ring degradation in the periplasmic space.

Tetracycline binds to 30S subunit of the ribosome to prevent ribosome translocation. tet resistance produces a protein that prevents tetracycline from entering the cell.

Chloramphenicol binds to the 50S subunit of the ribosome to prevent protein synthesis. cat (chloramphenicol resistance) produces an enzyme system component that converts chloramphenicol to a form that cannot bind the ribosome.

Kanamycin (and the closely related neomycin) are aminoglycosides that bind sub-components of the ribosome and prevent protein synthesis. kan (and neo) resistance depend upon the synthesis of an aminoglycoside phosphotransferase located in the periplasmic space that inhibits their transport into the cell.

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.

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.

Elements for expression

A cloning vector need not contain suitable elements for the expression of a cloned target gene; 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.

Types of cloning vectors

The antibiotic resistance genes encoded by plasmid DNA are often used in the construction of vectors for genetic engineering, as they provide means of selecting cells containing the plasmid.

Inserts larger than 20 kb are lost easily in the bacterial cell.

The pBR322 plasmid is one of the first plasmids widely used as a cloning vector



Though they are in many ways more specialized than plasmid vectors, they fulfill essentially the same function (act as carrier molecules for fragment DNA).

Bacteriophages fall into three main groups: i. Tailless, ii. Head with tail, and iii. Filamentous.

The genetic material may be single or double-stranded DNA or RNA. In tailless and tailed phages the genome is encapsulated in a protein shell called capsid.

Phages may be either virulent or temperate, depending on their life cycles. When a phage enters a bacterial cell it can produce more phages and kill the cell (Called the lytic growth cycle), or it can integrate into the chromosome and remain in a quiescent state without killing the cell (lysogenic cycle).

Virulent phages exhibit a lytic life cycle only. Temperate phages exhibit lysogenic life cycle, but most can also undergo the lytic response when conditions are suitable,

For example the lambda (45kb) phage contains a central region of 15 kb that is not required for replication or formation of progeny phage in E. coli. Thus, lambda can be used as a cloning vector by replacing the central 15 kb with 10-15 kb of foreign DNA.



- The bacterial artificial chromosome's usual insert size is 150-350 kb. A similar cloning vector called a PAC has also been produced from the bacterial P1-plasmid.

- By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of Bacterial artificial chromosomes (BAC).

- The primary components of a YAC are the autonomously replicating sequence (ARS), centromere, and telomeres from S. cerevisiae.

- Since they are maintained in yeast (a eukaryote), they are useful for cloning eukaryotic genes that contain introns. Also, eukaryotic genes are more easily expressed in a eukaryotic host such as yeast.

- Additionally, selectable marker genes, such as antibiotic resistance and a visible marker, are utilized to select transformed yeast cells. Without these sequences, the chromosome will not be stable during extracellular replication, and would not be distinguishable from colonies without the vector.

- Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic proteins that require posttranslational modification.

- 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.

Hosts for cloning vectors

1. prokaryotic cells

- Have space for foreign inserts.

- Have unique restriction sites for common restriction enzymes.

- Have convenient markers for selection of transformants, e.g. antibiotic resistance genes.

- Be relaxed, i.e. multiple copies in a host cell.

2. Yeast cells

3. Plants cells

Overview of Recombinant DNA Procedures



into their own chromosomes by recombination. However, many bacterial cells are unable to spontaneously transform. They first must be treated with chemicals to make them “competent,” or able to take up external rDNA.

For example: Calcium chloride (CaCl2) transformation is a laboratory technique in prokaryotic (bacterial) cell biology. It increases the ability of a prokaryotic cell to incorporate foreign or plasmid DNA allowing them to be genetically transformed. The addition of calcium chloride to a cell suspension promotes the binding of DNA to lipopolysaccharides (LPS). Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core. The DNA can then pass into the cell upon heat shock, where chilled cells (+4 degrees Celsius) are heated to a higher temperature (+42 degrees Celsius) for a short time.

2) Electroporation

Cells with walls must first be converted to protoplasts or spheroplasts (G+ and G- cells, respectively, without their cell walls) for this technique to work.

3) Gene gun

Microscopic particles of gold or tungsten are coated with DNA and shot out of a gene

gun with a burst of helium; the particles can penetrate plant cell walls and enter into plant cells.

4) Microinjection

A glass micropipette/ or a tiny needle is used to inject DNA into a cell.

NB: The inserted DNA, regardless of method, must be on a self-replicating plasmid or integrated into the host genome - if not it will be degraded and lost from the cell.

NB: For illustration of the process, see the figure below;



Selecting a clone with the gene of interest (Screening of clones) using blue-white screening method

Introduction

Rationale:

a) If the gene of interest codes for the production of an identifiable product, the bacterial isolate only needs to be grown in culture and tested.

b) Otherwise, the gene itself must be identified in the bacterium via other procedures.

Identification of the specific gene of interest in a clone library

A. Probing for the gene

i. DNA probe

- DNA probes are based on the fact that a denatured (heated or chemically treated to become single stranded) DNA molecule will hybridize (bind) to sequences that match or are similar to it.

- But where does the probe DNA come from?

C. Tagging


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