Expression Hosts and Vectors in Eukaryotes | Cell Biology

The below mentioned article provides an overview on Expression Hosts and Vectors in Eukaryotes.

One of the main objectives of gene cloning is to note the expression of the cloned gene encoding the protein. It is generally done in a foreign organism, for producing at a high level and again some hosts are needed for this purpose. These vectors are called expression hosts. The expression hosts fall under a number of bacteria, yeast and some mammalian cell lines.

Most of the works in this line have been done in E. coli, some species of Bacillus in having a property of producing some excretory proteins, yeasts, (Saccharomyces cerevisiae), have also been used as some post-translational modifications of some mammalian genes can be done.

Some filamentous fungi and actinomycetes are also being used for this purpose. The application of expression hosts has been widely used in pharmaceutical industry.

Expression Vectors:

When a gene or DNA fragment with entire structural gene including the promoter is in­serted into a vector, then the expression of that gene located in the vector, even if it is from different organisms, occurs in an organism (hosts—bacteria or yeast) after transformation.

Then these types of vectors are called expres­sion vectors—responsible both for gene expres­sion and gene cloning. For the purpose of gene expression, vectors are to be constructed; whose first requirement is the insertion of promoter along with the gene.

Some of the common promoters used for the construction of these vectors are the PGK promoter from Phosphoglycerate Kinase, GAL 1 from galactokinase, PHO 5 from Acid phosphatase and HSP 90 from a heat shock protein.

One such example of expression vector is PMA 91 (Fig. 17.25):

In this constructed vectors, the promoter consists of some sequence which codes for a few N-terminal amino acids of the protein that helps to initiate translation process. This has been used so that it does not interfere with the product of the desired gene.

This expression vector has been used for the production of im­portant proteins like interferon and thymosin in yeast cells. There are other expression vectors from E. coli and other bacteria.

But the use of expression vectors in the yeast system has some additional advantages than that of E. coli.

These are:

i. Like E. coli and some tumour cell lines, yeasts are not harmful to human being.

ii. Yeast does not produce any toxin sub­stances.

iii. Presence of inducible promoters, temperature-sensitive and other regulatory mutants help in regulating easily in the synthesis of proteins from the inserted gene.

iv. Yeast, being eukaryotic, is the better choice in producing active forms of eukaryotic proteins.

Use of Expression Vector in E. coli:

To obtain expression of eukaryotic gene in bac­teria, say E. coli, generally the gene is inserted in the downstream from the promoter. The most common promoter used in the bacterial system is the lac or trp operons. In this case the entire lac region and the first few nucleotides of the 0 galactosidase structural gene are inserted in the constructed vector for the initiation of translocation.

The foreign gene is then inserted downstream of some se­quence (N-terminal) of the β-galactosidase gene thus giving rise to a fusion gene. Thus the foreign gene comes under the control of the operon system of E. coli. One good example to produce a stable foreign protein in E. coli through fusion protein system is the production of Somatostatin, a peptide hormone.

After Somatostatin, a number of other proteins like insulin, thymosin, neo-endorphin etc.. have been produced in E. coli. Besides lac operon fusion genes have also been constructed with other bacterial genes as well.

DNA Sequencing:

The sequencing work was performed first on RNA, particularly tRNAs which were easy to obtain in sufficient quantities. The RNAs isolated were broken into small fragments and sequencing on each fragment was done through chromatography.

The first sequence of yeast tRNA was done is 1965 by Holley and others (Fig. 17.26). With the discovery of restriction enzymes and DNA polymerases, studies on DNA sequencing in large scale started around 1970. On the basis of primed synthesis of DNA, the first sequence of DNA of 5.4 Kb of bacte­riophage φX was done by Sanger and others in 1977 with the help of gel electrophoresis.

Two methods for quick sequencing of DNA were developed by F. Sanger and A. R. Coulson in 1975 and another by A. M. Maxam and W. Gilbert in 1977. In the first method, enzymes are used for sequencing DNA and the second method utilizes chemicals.

Sanger’s method uses a copying technique with a discontinuous primed DNA synthesis to get a small fragment which are then reacted with nucleoside phos­phates. Maxam and Gilbert’s method uses chemical degradation procedure to cleave the DNA. In both methods, DNA is labelled with p³² followed by autoradiography.

Sanger’s Dideoxy Method:

Sanger used dideoxy nucleoside triphosphates in his method of sequencing DNA. These are like the normal deoxynucleotide triphosphates, but the 3′ OH group is absent. When the normal deoxy group is present continued chain formation goes on during synthesis of DNA.

When dideoxynucleotide group is added in the reaction mixture, the reaction or DNA syn­thesis stops because the absence of 3′ OH group interferes with the normal synthesis of DNA (Fig. 17.27). In this method, DNA synthesis is done in the presence of four normal deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) along with a short primer.

After the synthesis of DNA, every time a dideoxy­nucleotide of one type out of the four types (ddATP, ddCTP, ddGTP and ddTTP) is in­corporated and immediately premature ter­mination of DNA synthesis occurs. In this way, a series of incomplete DNA fragments are produced which can be clearly demonstrated through agarose gel electrophoresis.

On the basis of the varying sizes of the fragments, bands at different loci were found. The separa­tion of template DNA through four dideoxynucleoside triphosphate treated samples can be easily analysed after autoradiography in the gel (Fig. 17.28).

Sanger’s method can be clearly explained with an example. Let the sequence of template DNA be 5′- A G T G A T C A -3′. When the template is allowed to synthesize, the new strand of DNA will beTCACTCGT.

Now dideoxynucleotides are added at different time, then the incomplete fragments of the template will be:

These fragments differ from one another in length by one nucleotide. Thus these fragments will form bands at different loci in the gel. The smaller fragment, i.e., with 2 nucleotides here, will be at the bottom.

The sequence of the DNA can be directly read from the gel:

This method was used to make breakthrough in sequencing the 16.5 Kb human mitochondria genome by Anderson and others in 1981.

Maxam and Gilbert’s Method:

The most commonly used technique for sequencing of DNA was developed by Allan Maxam and Walter Gilbert in 1977. This technique is mainly based on the chemical DNA sequencing method, as here chemicals are used for sequencing rather than enzymes.

The main principle of this method is that four chemical reagents are used to cleave single-stranded DNA chains near the specific bases, such as:

(1) Guanine,

(2) Cytosine,

(3) Adenine and Guanine and

(4) Cytosine and Thymine.

Before cleaving this DNA, the 5′ end of single-stranded DNA is labelled with p³². These fragments are then identified through autoradiography after their electrophoretic separation.

The detailed method is:

i. Cloned DNA is cleaved by an appropriate restriction enzyme to produce fragments with unique ends.

ii. The fragments are then run on agarose by electrophoresis.

iii. DNA fragments are then eluted from the gel.

iv. Ends of the fragments are labelled with p³² using DNA polymerase I, whose both ends are labelled.

v. Labelled double-stranded DNA is to be converted to single-strands by denaturing with NaOH and single-strands are isolated on a neutral gel via electrophoresis.

vi. Secondary digestion by some specific re­striction enzyme is done to cleave the fragments between ends after elution.

vii. Then chemical treatment is done to modify one or more bases of DNA through substi­tution of Purine or Pyrimidine ring.

viii. Four fractions for A, G, C -H T and T can be isolated.

ix. Each fraction is treated with some chemi­cals to destabilize the Phosphodiester link­age.

For the base Adenine (A), NaOH and EDTA solution at 90°C are used.

For G:

Dimethyl sulphate at 20° C is used.

For C+T:

Hydrastinine at 20°C is used.

For T:

Hydrazine and NaCl at 20°C are used for modification of the bases.

x. Cleavage of the nucleotides containing modified bases is done by heating the solution in 1M Piperidine solution at 90°C for 30 minutes.

xi. These fragments are then run on agarose gel. The larger fragments migrate slower than the smaller ones. The electrophoresis process takes about 30 hours to complete the run. The slab gel is then covered with plastic sheet (Saran) and X-ray film is placed on the plastic and is kept in the dark for exposure, p³² labelling will show some dark streaks in the lane.

To understand the method of DNA sequenc­ing, let the sequence of a fragment to be sequenced is

In this sequence, Guanine (G) is present in the second, fifth and tenth position. The chemical cleavage reactions for Guanine will produce fragments of 1, 4 and 9 bases with one end labelled with p³² if the fragment is radioactively labelled. On electrophoresis, these fragments showed 3 bands when the fragments are cleaved at position G. Similarly, other chemical cleavage reactions in other bases can be analysed (Fig. 17.29).

Due to non­-availability of single-stranded templates and primers, the efficiency of Sanger’s method was low at the beginning. Recently, these problems have been solved with the development of M13 cloning vectors and oligonucleotide synthesizer. Maxam and Gilbert’s method was employed to know the complete sequence of the 40 Kb bacteriophage T7 by Dunn and Studier in 1983.

These methods help to develop the modern au­tomated sequencing technology using dideoxy method recent automated fluorescent sequenc­ing instrument has developed. Various types of automated sequences, automated gel elec­trophoresis, raw data acquisitions, computer-operated robotic work stations and sophisti­cated software’s have enhanced the efficiency in handling sequencing reactions.

The application of Polymerase chain reaction (PCR) has accel­erated the sequencing studies to a great extent.

The use of PCR and dideoxy method has made possible in the development of cycle sequencing which can amplify the very small detectable signals—thus increasing the efficiency of se­quencing technology. The addition of single- strand binding proteins to sequencing reactions has improved the quality of the data.

Free Flow Electrophoretic Procedure:

This is a well-known and recent technique using free flow electrophoresis instrument which is of considerable help in recombinant research. This instrument is used for the separation of different micro and macromolecules, cell or­ganelles etc. on the basis of differences in electrophoretic mobility. The whole process is under completely automated stage.

This is similar to gel electrophoresis but there is an open flow separation chamber. The solution flows down perpendicular to an electric field. The sample is injected at the starting point of the flow and the separation of solutes occur in an angular direction.

The run-time of the sample is generally 6-12 hours. Con­tinuous reading of the flow is made through optical-electronic combining method. Using this flow-electrophoresis unit DNA fragments can be separated more precisely than through gel electrophoresis.

Automatic DNA Sequencing:

DNA sequencing can be divided into two types on the basis of fluorescent dyes used during processing. The first one is the automated sequences of single colour, 4 lane loading type (Pharmacia ALF, L1-COR 4000 of Millipore Base Station) with 10 or 12 channel capacity.

The second type is four colour, single lane loading (e.g., ABI 373A sequences) with 36 channel capacity per run.

The main principle of this automated se­quence is to use dye either as:

(1) Dye Primers, or as

(2) Dye-terminator.

(i) Dye Primer:

In this process, four separate reactions are done for each sample using different dye-labelled primer. They are then mixed and loaded in a single channel before electrophoresis. Taq poly­merase or T7 DNA polymerase (Sequenase) can be used in this system. As the sequence is amplified, so very small sample like 0.1-0.2 fig of template can be used.

Sometimes the standard dGTP is replaced by deaza-dGTP to avoid the gel-compression problem. In analysing large number of samples in a short time, a robotic system of ABI catalyst 800 or the Beckman Biomek 1,000 are used. The first robot c£m handle 24 sequencing reactions within 4 hours.

(ii) Dye Terminators:

Another system is to add the fluorescent dyes to the position of di-de-oxy-terminators. In this case, each of the four dideoxy nucleotide triphosphates (ddNTP) is labelled with dif­ferent dye.

The most commonly used dye terminator is Rhodamine Dye which works well with Taq polymerase. In this case, any primer and a variety templates like single-stranded DNA, double-stranded DNA, PCR-generated fragments can be used.

Sometimes dGTP is replaced by DITP (deoxy inosine triphosphate) to avoid gel compression. This dye terminator system (Taq/dye terminator) is used to know the preliminary sequence data quickly where 100% accuracy is not necessary. However, the high reading accuracy (450 bp at 99%) can be obtained when dye primers are used.

Use of Capillary Electrophores in DNA Sequences:

To get a high resolution as well as maximum sequencing rate, capillary tubes of gel are used instead of normal slab gel. The thin-walled (50-150 µm) fused silica capillaries (20-200 um², internal diameter) are used for the rapid dissipation of heat and for saving separation times. Capillary electrophoresis also provides rapid, high field, high resolution separations with very small sample loads.

Large number of scientists are engaged on the refinement of capillary electrophoresis for DNA sequenc­ing and have developed several laser-excited fluorescence detection methods. Recently a laser-excited, confocal fluorescence scanner has been developed to detect bands on mini DNA sequencing gels.

In this scanner, the laser is focused on the sample by an objective which is an ideal system for scanning arrays of micro- capillaries. It has the ability to detect multiple capillaries in parallel and these separations are 10 times faster than typical slab gels. One colour or two colour scanners can be used in the capillary array electrophoretic (CAE) apparatus.

The main part of this instrument is the laser- excited confocal fluorescence capillary array scanner with an argon laser beam (excitation at 488 nm). The beam is reflected by a beam splitter after passing through a 32X, 0.4 N.A. objective.

It is then brought to a 10 /mn diame­ter focus within the fine capillaries in the array. The fluorescence is then sent back through the first and second beam splitter separating the red and green channels. The beams are then passed through various filters for photomultiplier detection.

The computer-controlled stage is used to scan the different capillary arrays. The image and electrophorogram patterns were done with the help of Canvas and Kaleidograph programmes.

Capillary array photograph is obtained by scanning of a 24-capillary array containing the fluorescent primer labelled mixture of M13 mp 18 T-sequencing fragments. The bands are well resolved to detect large number of bands (Fig. 17.30). This CAE method is useful for high-speed DNA sequencing of PCR-amplified products.

Sequencing by Hybridisation (SBH):

Besides Sanger’s method, another method is recently been developed which is known as sequencing by nucleic acid hybridisation.

The main principle of this technique is the deter­mination of the sequence of unknown DNA fragment with a series of known DNA probes, with the successful hybridisation of short oligo­nucleotides, sequencing of many DNA frag­ments including 340 bp has been determined by Dramanae and others, 1993.

Now it has been established from several experiments that SBH have an important role in the sequencing of hu­man and other biologically important genomes.

The method of sequencing by hybridisation can be explained by the following example:

A 12 mer DNA target (A G C C T A G C T G A A) is mixed with different octamer probes. Suppose these probes have the sequences of TCGGATCG, CGGATCGA, GGATCGAC, GATCGACT and ATCGACTT. If there is perfect complementary sequence in the target DNA, then these probes will hybridize. Here the DNA sequence is thought of as an overlapping oligonucleotide sequences.

If this is so, then the alignment of the overlapping sequence from the hybridizing probes will help in the reconstruction of the sequence of the target DNA as follows:

The continuous sequence complementary to the analysed strand can be derived if the probes read vertically. Hence the sequence will be

5′-T C G G A T C GA C T T – 3′.

The reconstructed target will be

5′- A G C C T A G C T G A A – 3′.

This type of approach, called the finger print­ing approach, is the most efficient method for sequencing DNAs. SBH is. carried out by fixing the target DNA to a surface which is then mixed with different oligonucleotide probes, one at a time. This method can also be carried out by attaching probes to a surface, then the target DNA is added to the array of probes.

Two ways of sequencing by hybridisation are possible such as:

a. based on array of DNAs, and

b. based on oligonucleotides.

In the first way, labelled oligomers and, in the latter, labelled DNA fragments have to be used. The first process is suitable for large scale mapping and sequencing and the latter for the sequencing of short DNA fragments and for diagnostic purposes. Another method

of DNA sequencing is the Fluorescent Single Molecule Sequencing. The principle of this method is that a single DNA fragment is fixed on a magnetic bead in a flowing solution. The enzyme exonuclease is then added which acts on the DNA releasing one base at a time.

The released base is then identified as it flows through a laser-induced fluorescence detector. Generally the fluorescent dye Rhodamine is used in aqueous, ethanol and other solutions.

Dot and Slot Hybridisation:

Dot hybridisation methods were first estab­lished for the quantitative estimation of DNA or RNA by spotting a small amount of nucleic acid on dry nitrocellulose or nylon membrane. This was then hybridized with a specific DNA or RNA probe labelled with p³². The membrane was then exposed to X-ray film. Through this procedure the amount of RNA or DNA present in a population can be determined.

This method is very fast and sensitive and the quantitative estimation can be very easily done through Liquid Scintillation Counting. Again, the densitometric scanning of the X-ray film can be made after developing the film to note the amount of RNA/DNA or the rate of hybridisation of the nucleic acids.

The probes used in this dot/slot blot method (Fig. 17.31) can be prepared through different methods like nick translation, random primed hexanucleotide synthesis method etc.

Nick Translation:

This is a method for labelling a double-stranded DNA molecule with radioactive compound us­ing DNAase I and DNA polymerase I. The term Nick means the single-stranded gaps in the backbone of the DNA molecule.

When DNA is incubated with the enzyme DNAase I, DNA polymerase I and the four deoxynucleoside triphosphates like dA TP, dGTP, dCTP and dTTP, then the double-stranded DNA is nicked with DNAase I opening a site for labelling with radioactive source provided one of these triphosphates contain p³² in the phos­phate group.

DNA polymerase will repair the nick after the incorporation of deoxynucleotide triphosphates to the 3’OH group. DNA poly­merase also removes 5′ nucleotide of the nick due to its 5′-3′ exonuclease activity. Then the free nucleotides are replaced in these gaps with at least one nucleotide triphosphates as labelled with p³² (Fig. 17.32).

For example, after exonuclease activity, if G nucleotide is left on the intact strand, then the new DNA strand will be labelled with p³² labelled cytosine (C). In this way DNA becomes labelled with p³². Then this DNA is denatured to produce single- stranded molecules. Now this single-stranded labelled DNA fragments of 5-20 bases can be used as a probe.

Random Primed Synthesis:

In this method, short oligonucleotide primer is annealed on a single-stranded DNA strand. With the help of a DNA polymerase lacking a 5′-3′ exonuclease activity, DNA synthesis can be done over an intact single-stranded DNA from the primer.

These primers are generally a mixture of random hexanucleotides which are labelled generally with a p³² dNTP. The size of the templates should be more than 200 bp in order to get high activity and low background during hybridisation. Sometimes DNA synthe­sis can be started from an oligonucleotide of known sequence.

Use of Riboprobes for Labelling RNA:

The synthesis of RNA takes place on a DNA template with the help of RNA polymerase and ribonucleoside triphosphates. If some ra­diolabeled ribonucleosides are used during the synthesis of RNA, newly synthesised molecules will be labelled.

This labelled RNA transcript is known as riboprobes. There are large number of plastids for producing Riboprobes. Some of the commonly dsed vectors are pBS SK⁺ with bacteriophage RNA polymerase promoters T3 and T7; pGEM-3Z with SP6 and T7 promoters; pSPORT 1 with SP6 and T7 promoters etc.

The method of introducing a DNA fragment into a vector for the synthesis of RNA tran­script has now been changed with the devel­opment of Polymerase Chain Reaction tech­nology (PCR). In the case, RNA polymerase is to be added in the PCR reaction.

Before the transcription, the plasmid DNA is to be liberalized in order to avoid in the production of RNA transcripts from the plasmid DNA. This method is used to produce RNA tran­scripts which can be used to synthesize sense or antisense probes.

Assessment of Labeling Intensity After Hybridisation:

This includes the use of X-ray film over the gel or membrane blot producing bands correspond­ing to the location of the radioactivity. But the assessment from the autoradiography depends on the nature of the isotope. Isotopes p³² I¹²⁵ have strong emissions, so few are absorbed in the X-ray film and the rest passes through the film.

In this case an intensifying screen is placed behind the X-ray film in order to get good radioactive bands. This is known as indirect autoradiography. The intensifying screens containing calcium tungstate particles emits may flashes of light when radioactive particles fall on it. So a photographic image is produced over the auto radiographic image and thus giving the very clear bands.

Again there are some isotopes which have very weaker emissions such as H³,p³³,S³⁵ and C¹⁴. Labelled samples with weaker isotopes cannot be detected through autoradiography, so some fluorography techniques are used in order to detect the labelling. For fluorography, the gel is incubated with a scintillating fluid (PPO) to increase the sensitivity of the film.

The properties of different isotopes used for labelling are given in Table 17.6.

Non-Radioactive Lcalisation of DNA or RNA on Mmbrane:

The main principle of this method of localisa­tion is of using chemiluminescence through the conjugation of biotin to the protein Avidin or Streptavidin.

This conjugation can be identi­fied by using Avidin as epitope site for anti-avidin secondary antibodies with some reporter enzymes like horse-radish peroxidase (HRP) or alkaline phosphatase. Biotin can be attached to nucleic acids (DNA or RNA) through a process called Biotinylation.

This biotinylation can be done enzymatically by using biotin-labelled nucleotides or by adding chemically a long chain of biotin with some oligonucleotide through photo-biotinylation. Hybridisation of bio-tinylated nucleic acids (as a probe) to the nucleic acids on a membrane blot can be identified by staining or chemiluminescence (Fig. 17.33).

The biotinylated system with HRP as a reporter enzyme is known as Enhanced chemiluminescence.

Some staining methods for the detection of biotin-labelled nucleic acids are given in Table 17.7.:

 

Polymerase Cain Raction (PCR):

In 1987, a procedure was developed for the amplification of DNA templates which was designated as the Polymerase Chain Reaction or PCR in short. It is now found to be a revolutionary technique in Molecular Biology.

Principles of PCR Technology:

It is based on the synthesis of DNA by the enzyme DNA polymerase, separating the new strand produced and the ability to synthesise the strand over and over again—to amplify a single molecule of DNA until sufficient amount is produced to detect by conventional methods.

PCR Reactions:

These reactions can be carried out in 0.5 ml microfuge tubes using thermal cycler or temperature cycler.

The ingredients required for the reactions are:

(1) A Template DNA,

(2) Primers,

(3) DNA polymerase,

(4) Buffer solution,

(5) dNTPS.

Sometimes 10% glycerol and 5% DMSO (Dimethyl Sulphoxide) are added to increase the efficiency of PCR reactions (Fig. 17.34).

1. DNA Template:

The template DNA may be bacterial DNA, other genomic DNA (50-100 ng), Sperm DNA (5-10 µg). Sometimes RNA is also used as a template, and, in that case, reverse transcriptase is to be used.

2. Primers:

There primers are generally short single-stranded DNA of 16-35 bases in length.

These are some precautions in selecting the short DNA sequence as primers:

(a) Primers must have no complementary to each other.

(b) Primers should not form secondary struc­tures within their sequence (Fig. 17.35).

(c) 3′ end of the primer must have complemen­tary sequence to that of template DNA.

(d) Each primer pair must have same GC content so that annealing temperatures remain same.

The concentration of primers may vary but generally 10-100p mol is used in most of the PCR systems.

3. DNA Polymerase:

The DNA poly­merase used in the PCR systems is Taq polymerase, originally isolated from the thermophilic bacteria, Thermus aquaticus. This Taq polymerase can withstand higher temperatures which are necessary for the denaturation of DNA in the thermal cycle. Several heat stable DNA polymerases are now available in the market besides Taq polymerase (Table 17.8).

4. Buffers:

Reaction buffers are generally supplied along with the polymerase enzymes by the commercial firms.

However, the following buffer components are found to show good result for amplification of DNA: Standard PCR buffer without Mg++ (Kit from Perkin-Elmer/cetus), 3µl100 mM Mg⁺⁺ (3mM final concentration), 100 ng primer, 200 fiM each dNTP final concentration, 0.5 µl Taq (5 Units/µl), 10 µl of Template (from Triton stock) were brought up to 100 /d with double distilled water.

5. dNTPs:

Deoxynucleoside triphosphates of the four bases are also available from the suppliers.

Procedure:

The procedures of the PCR systems may be divided into three steps, such as Template de­naturation, Primer annealing and Chain elon­gation or extension.

First Step:

In this step, starting DNA molecule, i.e., the template DNA, is denatured by raising the temperature to a point where the hydrogen bonds holding the double strand structure of DNA are disrupted. The temperature varies between 92°C and 97°C.

Templates with high GC content require higher temperature in de­naturation of the strand. Large templates (1.5- 5 Kbp) also require higher temperature as the Tm (melting temperature) value is high. For better efficiency, reaction mixture is heated to 95° C for 5 minutes before adding Taq poly­merase.

Second Step:

Short single-stranded DNA molecules, called primers, are generally between 16 and 40 bp in length with exact matches of template se­quence. When the template sequence is not defined, then a mixture of primers are used.

The perfect matching of the primer with tem­plate DNA require correct annealing temper­ature. Perfect matching primers generally require annealing temperature of 55°C-72°C whereas mixture of primers with some random nucleotides (degenerate primers) require 45°C- 55° C for annealing.

The annealing temperature of the primer can be calculated on the basis of AT/GC pairing. For example, the annealing temperature is 2°C per AT/AU pairings and 3°C per G.C. pairings. If the sequence of the primer is G G C C A T T C A C C A C C T T T G G G G C C C C, there are 17 G and C and 8 T and A bases, hence the initial annealing temperature will be

Generally, the primers are added in excess, so that they anneal with the templates faster than the two templates can anneal with each other.

Third Step:

This is the chain extension process which is carried out at 72° C. The time required for the synthesis of new strand by DNA polymerase depends on the length of the region to be copied. The activity of Taq polymerase is assumed to be 200 bases/sec. These three steps generally require generally temperatures of 94°C,55°C and 70°C.

As the Taq polymerase can withstand high temperature, this process of reaction can be repeated; in other words, there are more cycles. The number of cycles may vary between 20 and 60. This shows amplification process increases with the increase of cycles. But it declines after certain cycles and then flattens out (Fig. 17.37).

PCR amplification can be rapidly scanned by running 10% of the reaction on 1-2% agarose minigel at 100V for 1h. The number of template molecules becomes double with each cycle, i.e., 2 at the end of first cycle, 4 at the end of second cycle, then 8, 16, 32 and so on.

PCR techniques is very useful but with some limitations in certain parameters like primer design, Mg⁺⁺ concentration, annealing temperature, and chain extension time. An­other difficulty is found is sequencing through the poly (A) tail of the cDNA in PCR systems which may be due to the slippage associated with PCR amplification of mononucleotides (poly-A) and di-nucleotides.

Applications of PCR Technique:

There are various applications of PCR in gene cloning such as:

i. Addition of some desirable ends to DNA fragments: This is done by us­ing some specially constructed oligonu­cleotides containing the desired end se­quences.

ii. Specific base alteration in a sequence through incorporation of mismatches into the sequence of the primer.

iii. Production of single-stranded DNA by keeping the primers at a concentration resulting in the production of single- stranded DNA which is used for sequencing or for the analysis of interactions of DNA and protein (foot printing).

iv. Cloning of unknown sequences: If the unknown sequences are placed at the end of known sequences of the primer, then the unknown sequences can be synthesised.

However, the synthesis of DNA through PCR is characterised by high rate of error in the incorporation of bases (about 0.25%), so any DNA fragments synthesised by PCR should be confirmed by sequencing.

DNA Finger-Printing:

With the help of restriction enzymes, a map can be established in DNA almost similar to genetic map which is called Restriction map. It differs from the genetic map in that any sequence of DNA can be mapped whether there occurs any mutation or not. But the genetic map locates the sites where there are mutations. The restriction sites or maps can identify if there is any base alterations or muta­tions.

The difference in the restriction map be­tween two species or varieties is called Restric­tion Fragment Length Polymorphism (RFLP). This RFLP can be used as a specific marker in the identification as well as to draw the phyto-genetic relationship between the species (Fig. 17.38). It shows Mendelian segregations.

The study of RFLP has been used for the identification of many genetic diseases and also in the forensic science. The identification of the altered gene in many genetic diseases and with the refinement of technology in synthesizing and cloning genes have led to the possibility of correcting the genetic defect by introduc­ing the normal gene in the patient which is known as Gdne therapy.

Researches are going on to introduce normal ,3-globiri gene to the thalassemia patient for the correction of the genetic defect.

The restriction analysis is also being used to identify individuals which has been known as DNA Finger-printing. With the progress of restriction analysis, the forensic scientists are now able to identify the criminals or murderers, disputed paternity, immigration cases etc. from blood and body fluid samples.

The use of hyper variable regions (HVRs) of human DNA consisting of some tandem repeats of short nucleotide sequences of 15-30 bp in length has opened a new vista in forensic science for the identification of individuals.

The length of DNA is variable in this region in the popu­lation. DNA probes are made from this region and are hybridized with the Southern blots of human DNA. The pattern obtained from this hybridisation is very individual-specific which is known as DNA fingerprint.

RNA Finger-Printing:

This method is used to note the sequence similarities between different species of RNA (Fig. 17.39). RNA fingerprints are based on the analysis of two-dimensional gels after partial digestion with ribonucleases. There are differ­ent RNAases which have certain specificity in digesting the RNA (Table 17.9).

 

 

Two-dimensional electrophoresis (2D) is a special technique to separate complex nucleic acid mixtures which cannot be clearly shown in one-dimensional gel. Polyacrylamide gels are used and the shift in the electrophoretic con­ditions in the two-dimensional gels is made by changing the acrylamide concentration, change from non-denaturing to denaturing or by chang­ing the pH.

Change in Acrylamide Concentration:

These changes separate the RNA molecules with different conformations in gel. Generally, the concentration of Acrylamide in the second dimension remains twice of the first. This method can separate RNAs which vary in size between 80 and 400 nucleotides.

Change From Non-Denaturing to Denaturing Condition:

Urea is used to initiate the denaturing con­ditions. This method helps to separate the RNA molecules that have hidden nicks in their structure.

Change of pH From pH 3.3 to pH 8.0-8.3:

In this method RNAs are separated on the basis of base compositions. The different bases, A, C, G and U, show different changes to the net charge of a RNA molecule at the acidic pH. The first dimensional gel is then placed into a gel mould where the second-dimensional gel is poured.

For RNA fingerprints, RNAase digestion is made first followed by two-dimensional analysis through gel electrophoresis or DEAE paper electrophoresis. RNA fragments are analysed in first-dimension gel on the basis of charge and in the second dimension on the basis of size.

RNA fingerprinting technique is used for the analysis of tRNA genes and their transcripts and also to study the viral genomes. The fingerprint analysis can be made on the basis of length and base composition of RNA.

In the first dimension, the base U present in the nucleotide shows high mobility and the order of decrease in mobility is found with the presence of G, A and C. In the second dimension, mobility depends on the size of oligonucleotides. Again the nature of these fragments can be confirmed through sequencing.

Gene Targeting:

It is a direct intervention at the molecular level to change the information contained in the DNA for obtaining some desirable characters. Conventional breeding method was an indirect method in manipulating genes.

There are three main criteria in the process of gene targeting, such as:

i. The process must be directed at the spe­cific locus;

ii. The process will help to insert a predeter­mined sequence at the target locus;

iii. The process should be efficient and repro­ducible.

The object of Gene targeting is the in vitro manipulation of DNA molecules and then the incorporation of these segments into the genomes of living cells.

The first step in this process is the isolation of mutants. We know that the mutation may be detrimental, neutral or beneficial. From the beneficial mutants, DNA can be isolated, sequenced, the fragments can be rearranged, cloned and then can be inserted into the genome of an organism.

In this way, inborn errors of metabolism can be corrected in human and the technique of gene therapy can be used for the cure of several incurable diseases. Some Gene therapy are already being applied to human. The method of Gene targeting was first experimentally shown in Yeast in 1980.

Later, it was also established in mammalian tissue culture cells. The main objective of gene targeting was not to insert the exogenous DNA but to inherit these DNA in the subsequent generations. For these, a suitable vector is used as in other genetic engineering methods.

But to serve the purpose of inheritance, a sequence for partitioning—which is recognised by the spindle apparatus—is introduced in the DNA molecule.

This partitioning sequence is present in the centromere. If there is no partitioning sequence, then the inheritance of the DNA molecule becomes a matter of chance. The marker gene necessary for the selection of recombinant molecule is already present in the constructed vector.

In case of yeast the common marker used is the LEU2 gene, which codes for the enzyme needed for the biosynthetic pathway of leucine. Leu 2^– cells require Leucine in the culture media for growth. An­other well-known marker in the yeast is URA 3 gene. Cells lacking URA 3 gene, i.e., URA 3^–, will require Uracil in the media for growth.

Another main criteria for gene targeting is the way to make homologous recombination between the vector and chromosomal sequences (genome). In other words, the foreign DNA fragments must contain sequences homologous to some part of the chromosomal DNA.

It has also been noted that these vectors have to be constructed without any origin of repli­cation which is known as Integrating vector (Fig. 17.40). This type of homologous re­combination is initiated with the development of a double-stranded break in the homologous sequence in the vector.

The insertion of exogenous DNA in the URA 3 gene or LEU 2 gene would result in the integration of DNA in the chromosomal URA 3 or LEU 2 gene. Thus the integrations can be targeted at specific sites of the chromosomes (Fig. 17.41).

The dominant genetic marker is used to target the exogenous genes showing gene targeting as a powerful tool in genetic engineering. The break in the genetic marker, say URA 3 or LEU 2 in yeast, is made prior to transfection. Then the desired mutation is constructed into the vector and then this mutation is inserted into the chromosomal gene URA 3 or LEU 2 by gene targeting prior to transfection.

There are four methods commonly used for gene targeting:

First Method:

This method is done through gene disruptions or knockouts. Here two independent mutations are introduced into gene say ‘X’ in the vector, for targeting. This mutation is done through small deletions at 5′ and 3′ ends and this mutation is separated to such a distance so that homologous cross-over can occur between them. The targeting is then done by cutting the vector in the middle of gene ‘X’.

Then the transfection is done into the yeast cells and the integration of the vector to the chromosomal gene^(x) is done. After a single reciprocal cross-over, the integration gives out a structure containing 2 copies of gene ‘X’ with the LEU 2 gene and vector sequences sandwiched in between.

Two copies of gene ‘X’ contain 5′ and 3′ deletion (mutation), respectively. There are many dis­advantages in this method, so this technique is not used now (Fig. 17.42).

Second Method:

In this method, gene ‘X’ is cleared in the middle and the marker gene LEU 2 is inserted in the space. Here also some loss of function of gene occurs due to the interruption of gene ‘X’. The targeting is done by introducing two double- stranded breaks on either side of the gene ‘X’.

Then the transfection is made into yeast cells. The inserted gene ‘X’ can be modified according to the need of the experiment. The selection can be made by collecting the LEU+ colonies. In this method, the targeted gene ‘X’ is stable and cannot revert to wild ‘X’ gene (Fig. 17.43).

Third and Fourth Methods:

These methods are used to introduce point mutations into the chromosomal gene. For making any experiment on targeting, the point mutation is inserted into the gene ‘X’ in the vector. These methods are also known as Gene replacement with a pop-in/pop-out targeting vector.

This is done by cutting gene ‘X’ in the vector keeping the point mutation on one side, then the transfection is made into the yeast cells and selection is done with the help of the marker (LEU 2).

The integration of the vector into the chromosomal gene takes place through single cross-over. Fig 17.43 B, one copy of gene ‘X’ contain point mutation while the other copy does not. During cross-over gene ‘X’ is aligned by forming a loop. This cross-over formation may form a structure with a gene ‘X’ without any point mutation or with point mutation.

This method is of wide use for the following reasons:

i. The mutation is stable.

ii. Targeted clones may have an unlimited spectrum of mutation.

iii. Chromosomal lucus remains unchanged.

Transgenic Mice:

Transgenic means the transfer of exogenous DNA in the genome of an organism and this type of organism is known as transgenic or­ganism. Transgenic organisms may be plant or animal. In order to obtain inheritance of the inserted gene or DNA, the integration of this DNA must be in the germ cells. For this, many experiments have been done in mouse using fertilised egg cells and embryonic stem cells (ES cells).

Nowadays, the mouse embryonic stem cells in culture are used for gene targeting experiments. The ES cells have one unique characteristic in that these cells can give rise to all types of cell, i.e., undergo differentiation.

In order to induce targeted mutation in mice, the mutation is induced in ES cells and then these are trans­mitted to the germ line by injecting the cells into an embryo. Hence the embryo carrying the mutated cells will produce transgenic mice. These mice are then bred to produce more animals (Fig. 17.44).

The most widely used method for the pro­duction of transgenic mice is by injecting the exogenous DNA into the zygote. DNA is then integrated into the chromosome. The egg then develops through mitosis and all somatic and germ cells of the transgenic mice have the new DNA.

However, the use of ES cells gradually becomes more and more popular in gene targeting experiment as the screening of ES cell clones is very easy as compared to the screening of transgenic animals (Fig. 17.45).

In case of gene targeting experiment, targeting vector is made as usual. Then DNA is isolated from the targeting vector and this DNA is incubated with ES cells for fusion using electroporation. After a certain period of incubation, DNA is introduced into the cell nuclei. The cells are then plated on dishes and then recombinant cells are selected using some marker (G 418^R) (Fig. 17.46).

In this way targeted clones made can be kept for long term storage under low temperature for future use. These ES cells are then injected into a host blastocyst (Fig. 17.47). The in­jected blastocyst is transferred to pseudopreg­nant females for the development of chimaeric animals. New offspring’s are then produced through breeding.

Thus gene targeting, i.e., the modification of genes in living organism, has a-great potential value in basic and applied research as well as in the correction of many genetic defects in human cells.