INTRODUCTION OF GENES USING PARTICLE BOMBARDMENT

11. INTRODUCTION OF GENES USING PARTICLE BOMBARDMENT

The initial steps in this experiment involved coating the gold particles with the DNA plasmid. 5 mg of gold beads were weighed into a centrifuge tube and 400 µl of 100% ethanol added. The solution was subjected to an ultrasonic disruptor 10 X per second and flashed at 10000 rpm. The supernatant was sucked off, and dried for 5 minutes under a clean bench. 4 µl of NaOAC and 40 µl of DNA (0.5 µg/µl TE) were added and the solution shaken vigorously. 100 µl ethanol was added and kept at –80 degrees C for 15 minutes. The solution was flashed at 10000 rpm at 4 degrees C and the sup was sucked off. 500 µl of ethanol (1 mgAu/100 EtOH) was added and the mixture subjected to ultrasonic disruptor 3 X per second, using a vortex, and gold beads solution added (10 µl X two times per plate). It was later dried up and bombardment done using a titanium coin.

The titanium coin was washed twice with double distilled water, then washed twice with 70% ethanol, and washed a third time with 100 % ethanol, and the gold particles solution applied on the coin and allowed to dry ready for bombardment.

SDS PAGE

9. SDS PAGE

This is a useful method to separate and/or identify proteins and nucleic acids, based on the polypeptide length to determine their molecular weight. The procedures used in the experiments were based on the Laemmli discontinuous gels. Initial steps involved the preparation of crude extracts from plant leaves (namely egg plant and tomato) where fresh leaves were chopped into small pieces and ground into fine powder with liquid nitrogen. The powder was melted, centrifuged at 15000 rpm for 15 minutes and supernatant transferred to a new tube and stored in ice.

The sample was prepared for SDS page by adding 15 µl of 4 x SDS buffer into a micro tube and 45 µl of crude extract, mixed well and mixture heated at 95 degrees C for 5 minutes.

The separating gel was prepared by assembling the glass plates and the acrylamide solution prepared in a 15 ml falcon tube, and mixed well by inversion. The solution was poured slowly into the spaces between the glass plates, ensuring that all bubbles were removed (if any), and a layer of small amount of water-saturated 2-butanol added carefully onto the surface of the gel solution, to prevent the inhibitory effect of oxygen for polymerization. The glass was subsequently covered with saran wrap to prevent drying.

Following steps included the removal of the overlayed butanol and using kimwipe to soak as much water as possible to by capillary action. The acrylamide solution for stacking the gel was prepared and mixed well by inversion, and then a clean comb was inserted into the gap of the plates thus making sample lanes. It was vital to avoid any air bubbles being trapped under the comb.

To run the electrophoresis, all the SDS-PAGE apparatus were assembled appropriately, comb carefully removed and the samples applied into the desired wells. The samples were ran at 15mA per gel for 90 minutes.

CBB staining was done to detect the proteins in the PAGE gel. In this experiment, a commercially available tool kit (ULTRA-FAST Coomassie Stain) was adopted. The gel was washed twice in 100 ml deionized water, and an adequate amount of staining solution added, and was shaken gently for 30 to 60 minutes. It was finally rinsed in large volumes of deionized water to enhance the intensity of protein bands.

INTRODUCTION OF GENES USING AGROBACTERIUM

8. INTRODUCTION OF GENES USING Agrobacterium

This lecture dealt with ways of introducing foreign genes in higher plants with a focus on the Tobacco transformation protocol. The initial stages of the experiments were preparation of Agrobacterium culture and the culture medium for transformation. YEB liquid medium was prepared and inoculated with Agrobacterium which was previously stored at –80 degrees C. The culture medium for transformation was also prepared namely co-cultivation media and selection media.

Tobacco leaf explants were isolated and cut into small pieces, placed on filter paper on the co-cultivation plate. The agrobacterium suspension was added to the leaf explants and leaves immersed for 10 minutes, and were later transferred to sterilized filter paper in a petri dish to remove the excess agrobacterium suspension. The explants were transferred onto filter paper on the co-cultivation medium and cultured for 2 days at 20-25 degrees C.

After the co-cultivation period, the leaf explants were transferred to the selection media aseptically under the laminar flow chamber. The explants were periodically sub-cultured after 2-3 weeks, and later observed of the transformed cells. These included regenerated shoots and calli that formed on the explants.

Any red-colored tissues that were observed were considered to have expressed the gene that was inserted.

DNA was isolated from the tobacco leaf tissues and a gel electrophoresis was ran at 100 volts for 20 minutes, and subsequently stained in Ethidium bromide molecules for 10-15 minutes for visualization.

This photo shows Electrophoresis of DNA extraction from transformed and non-transformed tobacco plants.

TREATMENT OF NUCLEIC ACIDS WITH ENZYMES

7. TREATMENT OF NUCLEIC ACIDS WITH ENZYMES

(Digestion of DNA with Restriction Endonucleases)

DNA digestion and ligation: chemicals and other reagents were prepared according to the following quantities and concentrations:

5 µl pUC 19 (0.5 ug/ul), 3 µl Hind III, 3 µl of 10 x M buffer, 17µl water and 2 µl bacterial alkaline phosphatase to give a total of 30 µl. Subsequently, a second solution was prepared consisting of 6 µl of 0.5ug/µl of lambda DNA was mixed with 3 µl Hind III, 3 µl of 10 x M buffer and 18 µl of water to give a total of 30 µl also. Both solutions were put in separate micro-centrifuge tubes, spinned down and incubated at 37 degrees C for 2-3 hours. The DNA fragements were checked by Agarose gel electrophoresis that was prepared by 1% gel in TAE buffer.

10 µl of the digestion mixture was picked, 5 µl of sample and 1 µl of loading dye, mixed and applied to the gel. The samples were spinned by centrifugation before application to the gel. Lambda DNA digested with Hind III was used as the molecular marker.

To recover the DNA from the gel, 25 µl of membrane binding solution was added to the reaction mixture and SV mini columns inserted into the collection tube. The mixture was transferred to the mini column assembly and incubated at room temperature for a minute. It was subsequently centrifuged for 1 minute at 15000 rpm, the flow through discarded and mini column re-inserted into the collection tube.

700 µl of membrane wash solution was added and spinned for 15000 rpm for a minute. The flow through was discarded and mini column re-inserted again into the collection tube. This was repeated again once. The mini column was carefully transferred to a clean 1.5 ml tube and 20 µl of water added. It was incubated at room temperature for a minute and spinned down for a minute aft 15000 rpm. The mini columns were then discarded and remaining tubes stored at 4 degrees C or at –20 degrees C.

SOUTHERN HYBRIDIZATION

6. SOUTHERN HYBRIDIZATION

This is a technique of detecting DNA by using a DNA probe. In this technique, DNA is separated by gel electrophoresis and then transferred from the gel to a membrane by blotting. The DNA was detected from the membrane with a DNA probe to complementary bind to DNA.The probe was labeled by Alkaline phosphatase, that was observed by chemical luminescence that was observed as a glowing light.

Digesting DNA with restriction enzymes, EcoRI was used, which recognizes the sequences of GAATTC and specifically cuts between G and A, and A and G of the complementary sequence.

In the experiments carried out, 2 fragments of 950 bp and 4550 bp respectively, were obtained.

Agarose gel electrophoresis: the agarose gel electrophoresis was prepared and a 6-well gel (for a larger volume) was used. 40 µl of sample and 8 µl of 6 x loading buffer (blue color) was loaded into each of the wells giving a total sample of 48 µl.

5 µl of 50ng/µl lambda Hind III was used as the molecular marker while the loading buffer used was 1 x TAE.

The DNA band was cut out of the gel under UV light and its weight taken. This was done by taking the weight of an empty eppendorf tube and repeating with the gel slice containing the DNA sequence inserted into the tube, and the difference calculated to obtain the actual weight of the gel slice. For each 100 mg of gel slice, 300 µl of buffer QG was added, hence for 178.24 g, 535 µl of the buffer was added. A second gel electrophoresis was carried out again.

Samples were loaded and ran for 30 minutes. The DNA was determined by using a restriction enzyme lambda Hind III for digestion.

Membrane neutralization: Pieces of positively charged nylon membranes were cut into 1 mm, and 6 sheets of thick blotting paper cut to the same size as that of the membrane. The corner of the membrane was cut to match that of the cut corner of the gel. 2 pieces of thick blotting paper were soaked with alkaline Denaturation solution on glass plate to form the support. The blotting paper was made wet with alkaline Denaturation solution and 3 pieces of blotting paper placed at the center of the support paper on the glass plate. Any bubbles that formed between the papers were removed. The top of the gel was made wet with alkaline Denaturation solution and wet membranes placed on top. A stack of paper towels (about 5 cm thick) were placed on the blotting papers and a glass plate placed on top of the stack and weighed down with a 300-g weight. This position was left for 8-24 hours to allow DNA to transfer, and paper towels were subsequently replaced as they became wet with buffer.

Hybridization: the paper towels and blotting papers were removed from above the gel, and gel laid upside down on a dry sheet of blotting paper. The positions of the gel slots were maked on the membrane using a soft lead pencil. The gel was peeled off from the membrane and discarded. The membrane was placed on the blotting paper soaked with neutralization buffer for 5 minutes; afterwhich it was placed on a new Saran wrap, and subsequently dried using a hair drier. Irradiation at 254 nm was done to cross-link the DNA to the membrane, and then wrapped loosely with aluminium foil or blotting paper. The membrane was finally stored at room temperature.

Preparation of labeled probe: 20 µl of crosslinker solution was diluted with 80 µl of water. DNA to be labeled was diluted to 10ng/µl concentration and 10µl of the diluted DNA solution placed in a microcentrifuge and denatured by heating for 5 minutes in boiling water bath and subsequent cooling in ice immediately for 5 minutes. It was spinned down, and 10µl reaction buffer added, mixed thoroughly, the 2 µl of labeling reagent added and mixed well but gently. 10 µl cross linker solution was added and briefly spinned down. The reaction was incubated for 30 minutes at 37 degrees C.

Post hybridization stringency washes were prepared for detection. A little primary buffer was put into a box and membrane placed into box, for washing. For the secondary wash, the second prepared buffer was added and membrane washed with gentle agitation. It was ensured that the membrane was maintained in wet condition.

A wrap film for placing the membrane was prepared, and membrane placed with the marked side up. 1.5 ml of CDP-star reagent was added on membrane and left to stand for more than 5 minutes, ready for detection under chemiluminescence.

DNA SEQUENCING

5. DNA SEQUENCING

This refers to the conversion of template DNA into small fragments. This is achieved through chemical methods and/or enzymatic reactions. The template DNA is denatured to single strands, where short oligonucleotides anneal to the single strands, (specifies the start of the DNA polymerization). Elongation subsequently follows and a specific terminator is added in the reaction. Primarily, there are 4 reactions for each ddATP, ddTTP, ddGTP, and ddCTP, and 4 lanes are available for each lane to measure the strands. This is termed as the classical way. In principle, the shortest fragment is positioned at the bottom/end of the plate.

It is noteworthy that for detection of the fragments, one must label them by use of fluorescent labeling using a fluorescent molecule, and/or by terminator modeling where a terminator nucleotide is labeled by a fluorescent molecule). In the former, each terminator can be labeled using 4 different color signals and one reaction can be done in one single tube; while the latter, one lane is sufficient for the reaction as each reaction is labeled.

Capillary electrophoresis: An alternative method is use of capillary (30-70 cm long) known as capillary electrophoresis, where a very high voltage is applied to have a faster flow of DNA and a detector is placed at the end of the capillary tube which automatically detects the color and reads simultaneously. This method requires 2 hours to read bases of up to 1000.

Cycle sequencing system: this system is similar to the PCR technique as the template is denatured and a primer molecule can use the same template to start elongation again. Elongation can be repetitive using a single primer only, and the template can be used many times. The double-stranded DNA can also be used as a template directly because they will eventually separate into single strands.

The experiments in this topic focused on:

1. The cycle reaction with 4 color terminator labels and
2. Capillary electrophoresis

Purification of the reaction products: this involved the precipitation of DNA fragments.

THE POLYMERASE CHAIN REACTION

4. THE POLYMERASE CHAIN REACTION

Basic PCR technique is important and indispensable in molecular biology, as the DNA fragment can be amplified. Useful in amplifying a DNA sequence whose nucleotide sequence is not known.

Principles of PCR are in 3 steps namely:

i) Denaturation at 94⁰ C (10-30 seconds) where one double-stranded DNA molecule is denatured to 2 molecules of single-stranded DNA.

ii) Annealing at 45-65⁰ C (30 seconds onwards depending on the size of the sequence) where specific primers can hybridize to the single strand in the opposite direction. In this step, if lower temperatures are set, the primer can hybridize many sites, while at 50-60⁰ C the primer can hybridize the target sequence only. Normally, amplifies 1kb/minute or 1 kb/30 seconds in modern PCR machines.

iii) Extension at 72⁰ C, the primer initiates to extend by use of DNA polymerase.

The above-mentioned 3 steps complete one cycle. In the second cycle, there are 2 double-stranded DNA that are denatured to single-stranded DNA to form 4 molecules of single-stranded DNA.

CASSETTE-LIGATION MEDIATED PCR

5 steps are involved in this kind of PCR namely:

1. Digestion of DNA with restriction enzymes by using Hind III.
2. Ligation of cleavage product to synthetic double-stranded DNA with Hind III site.
3. The first PCR using ligated DNA and template and 2 primers. One primer hybridizes to the target sequences and the other primer hybridizes to the cassette.
4. The second round PCR gives more specificity to amplification. The template is used from the first PCR product plus 2 primers. These 2 are engineered to be hybridized into the primer sites of the previous first PCR primers.

3’ – RACE ( 3 prime rapid amplification cDNA end)

This is used when cloning cDNA from eukaryotic cells such as fungi, we use the 3’-race method. In eukaryotic cells, mRNA are added to the polyA tail at the 3’ end. cDNA can then be synthesized using reverse transcriptase. Oligo dT primers are used in many cases to synthesis cDNA. Some sequences are also added at the end of the 5’ of the oligo dT primer. Then, the specific primer for the target sequence can be constructed, then a PCR can be done using the cDNA as the template. After the first round of PCR, a second round of PCR is done again; hence another primer must be constructed. Using this method, cDNA can be cloned rapidly. It is noteworthy that bacteria mRNA does not contain poly A tail.