Samples Collection - Genetics

Samples collection :

According to (154) with some modification in the method of collecting the aspiration fluid, the lesion sites were cleaned with 70% alcohol. there my a syringe (1ml,30G*1/2 )when intradermal pentostam injection at the periphery was done , we were aspirated the fluids and the blood that oozing from the sites by capillary tubes with anti-coagulants and prepared slide to examine directly by microscopy after staining and the other was saved in screw cap container with the Sterile NaCl solution as dilution fluid and incubated in refrigerator (4 c) until DNA extraction.

Microscopic Examination:

This examination conducted on each sample of aspiration prepared the smear by transferring a small portion of the sample onto the clean slide. Staining with Giemsa or Leishman's stain solution and the examined under the light microscope with a 100 objective lens. according to:

• Smears were used as a thin as possible and air dried.
• The smears were Fully covered with Leishman's stain solution for 2 minutes.
• twice the amount of distilled water was Added and mixed by swirling, incubated at least 10 min.
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• Rinsed thoroughly with distilled water.
• The slides dried using blotting paper and air dry.

Preparation showing the amastigotes is consider to be positive (+ ve ?) for the Lishmania spp. and preparation with no amastigotes is considered to be negative (-ve) for the Leishmania spp. All the results were recorded (125,154).

Molecular part :

DNA extraction :

The isolation of Leshmania spp. DNA was extracted from the aspiration fluid by using the (DNA extraction for the intracellular organism) Genomic DNA Mini Kit ( Geneaid , Taiwan ) according to the manufacturer's protocol and stored at _20oC (Appendix No.

2).

The components of the kit are as follows:-

Components Amount

• RBC Lysis Buffer 135 ml
• GT Buffer 30 ml
• GB Buffer 40 ml
• W1 Buffer 45 ml
• Wash Buffer (Add Ethanol) (100)ml
• Elution Buffer 30 ml
• GD column 100 pcs

2 ml collection Tube 200 pcs

(( Added absolute ethanol (see the bottle label for volume) to wash buffer prior to initial use ))

Genomic DNA Mini kit (Blood/ cultured cell) Protocol :

Step 1 / sample preparation :

• 300 ?l of a suspension of aspiration was transferred to 1.5 ml microcentrifuge tubes.
• Added 3X the sample volume of RBC Lysis Buffer then mix by inversion .do not vortex.
• The tube was incubated for 10 minutes at room temperature.
• Centrifuged for 5 minutes at 3000 xg then removed the supernatant completely.
• 100 ?l of RBC Lysis Buffer was added to re-suspend the leukocyte pellet then proceed with step 2.

Step 2 / cell lysis :

• 200 ?l of GB Buffer was added to the 1.5 ml microcentrifuge tube then shaked vigorously.
• Incubated at 60oC for at least 10 minutes to ensure the sample lysate is clear.
• During incubation, inverted the tube every 3 minutes.
• At this time, pre-heat the required Elution Buffer to 60oC. (for step 5 DNA Elution).

Step 3 / DNA Binding :

• 200 ?l of absolute ethanol was added to the sample lysate then immediately mix by shaking vigorously for 10 second Note: if precipitate appears .break it up as much possible with a pipette.
• The GD Column was Placed in 2 ml Collection tube then transferred all of the mixtures to the GD Column.
• Centrifuged suspension at 14 -16000 xg for 5 minutes.
• Discarded the 2 ml Collection tube then place the GD Column in new 2 ml Collection tube.

Step 4 / wash :

• 400 ?l of W1Buffer was added to the GD Column then centrifuged at 14 -16000 xg for 30-60 seconds.
• Discarded the flow-through the place the GD Column back in the 2 ml collection tube.
• 600 ?l of Wash Buffer (ethanol added ) was added to the GD Column.
• Centrifuged at 14-16000 xg for 30-60 seconds the discard the flow-through.
• The GD Column was Placed back in the 2 ml Collection tube.
• Centrifuged again for 3 minutes at 14-16000 xg to dry the column matrix.

Step 5 / DNA Elution :

• The dried GD Column was transferred to a clean 1.5ml microcentrifuge tube.
• 100 ?l of pre-heated Elution Buffer or TE was added to the centre of the column matrix.
• Incubated for 3 minutes at 37oC to ensuring Elution Buffer or TE was absorbed.
• Centrifuged at 14 -16000 xg for 30-60 seconds to elute the purified DNA, and stored the DNA at 2-8 oC.

DNA quantitation :

"DNA samples that prepared from the aspiration blood were quantified by the Ultraviolet spectrophotometer (Unico, USA) reading first at 260 and 280 nm (155). All the samples were stored at the ( -20 oC) until use."

The DNA for assessment was removed from cold storage for 30 minutes, flicking the bottom of tubes ensured that the entire DNA was suitably resuspended.

Samples were then centrifuged at 8000 xg for 5 minutes.

A blank was prepared by taking 1000 ?l of TE buffer and used to zero the spectrophotometer = first Quartz cuvette.

In the second Quartz cuvette, 10 ?l of the DNA sample was mixed with 990 ?l TE buffer (sample cuvette). After zeroing the spectrophotometer (Unico, USA) on the first cuvette was brought into place to obtain an absorbance reading.

"Reading was taken of wavelengths of 260 nm (OD260) for the DNA of the sample and the 280 nm (OD280) for detected the protein concentration of the sample; the spectrophotometer were re zeroed between each of the wavelength reading."

The ratio between the readings of the (OD260/OD280) it is provided to determination of the sample purity, which is should be become between (1-2). Values of (OD260/OD280) of the less than 1 indicate the contamination of the DNA by protein (Sambrook et al., 1989).

The concentration of the DNA (?g/?l ) were determined by using of the following equation:

"DNA Concentration (?g/?l ) = (reading in OD260) X (total/sample volume) X constant (50 ?g/1000 ?l ). & ?((OD in 260 nm x( dilution factor )x constant 50µg)/(1000µ²))"

The yield was estimated by (concentration X total volume).

DNA Quantitation by Gel Electrophoresis :

Electrophoresis of DNA extracted (Pre_PCR) on agarose gel was checked by illumination after staining with ethidium bromide as a method that showed by (156) with some slight modification. After staining, ultraviolet transillumination was used for illumination of the DNA bands in the gel.

agarose gel electrophoresis was adopted to confirm the presence and integrity of the extracted DNA (155).

Preparation of Electrophoresis Equipment:

Reagents used for electrophoresis :

Ethidium bromide.

Bromophenol blue in 1% glycerol (loading buffer).

Agarose.

• 1 X TBE buffer.
• PCR marker100bp ( Bioneer, Korea).

Preparation of agarose gel with ethidium bromide(Pre-PCR): (155).

• Aliquot of 1X TBE (100ml) was taken in a beaker
• Agarose powder (0.7g) was added to the buffer
• The solution was heated on Hotplate until all gel particles were completely dissolved and boiling until become clear. and left to cold down at 50-60 0C
• Ethidium bromide (2 ?l of 10mg/ml) was added to the agarose
• The agarose was stirred in order to be mixed to avoid making bubbles
• The solution was left to cool down at 40-45 0C

Procedure of Electrophoresis :

The casting of the horizontal agarose gel :

After sealing both edges of the gel tray with cellophane tapes and fixing the comb in 1 cm way from one edge, the agarose solution was poured into the gel tray, the agarose was allowed to solidify at room temperature for 30 min. The fixed comb was carefully removed and the gel tray was placed in the gel tank. The tank was filled with 1X TBE buffer until it reached 1-2 mm over the surface of the gel (155).

1. Six µ² of extracted DNA was mixed with 2µ² from loading dye then loaded on the well of the gel bucket.
2. The electrophoresis was carried out for about 1 hour with 50 volts depending on the distance between the cathode and anode in the electrophoresis apparatus according to the following equation: 5 volt/cm
3. When the electrophoresis was completed, the gel was placed on UV transilluminator.
4. A digital picture was made for evaluation and documentation of the results.

Polymerase Chain Reaction (PCR) :

"All suspension samples examined for the DNA extraction which was the assayed by the PCR amplification process. The specific all primers were synthesized from IDT (IDT, Inc ( USA ), and they were designed on the basis of the sequence information of the genes repeated the unit that amplifies a highly repeated the sequence of Cutaneous Leishmania DNA according to the following" :

Target genes of leishmaniasis :

ITS1 region :-

"The forward primer (LITSR) 5-CTGGATCATTTTCCGATG-3 and reverse primer (L5.8S) 5-TGATACCACTTATCGCACTT-3, specific to the ribosomal ITS1 region that occurs between the genes encoding the small subunit (18S) ribosomal RNA and 5.8S rRNA" according to (157,158), (appendix No.8).

mini-exone gene:-

"Amplification of the mini-exone genes performing as a single PCR with the forward (5'-TATTGGTATGCGAAACTTCCG-3') and the reverse (5'-ACAGAAACTGATACTTAT-AT AGCG-3') primers as the described in (120)", (Appendix No. 7).

kDNA:-

The kDNA PCR using the primers 13A (5'-GTG GGG GAG GGG CGT TCT-3') and 13B (5'-ATT TTC CAC CAA CCC CCA GTT-3') according to (159, 160), (Appendix No. 6).

Preparation of Primers :

All primers were supplied by IDT Company, these Primers (lyophilized product of different picomols concentrations) were resolved according to the instructions of the manufacturer, where resuspension using deionized water to Primer's tube, then placed in microcentrifuge with speed 8,000rpm for 1mintues for obtaining on stock solution with concentration 100 picomols, then was taken 10 µ² from stock solution in Eppendorf tube( 1.5 ml ) and 90µ² of deionized water was added for obtaining a working solution with a concentration of 10 picomols /?l of suspension.

Table(3.1): The preparation of the final concentration of the study PCR primers of cutaneous leishmania genes.

The final concentration of the stock solution (picomole/µ²) Concentration (picomole) Sequences Primer name

• 100.0 10.0 (LITSR) 5-CTG GAT CAT TTT CCG ATG-3 ITS1 (forward)
• 100.0 10.0 (L5.8S) 5'-GGT CAA GAG CTT ACA ACA CG-3' ITS1(Reverse)
• 100.0 10.0 (5'-TAT TGG TAT GCG AAA CTT CCG-3') mini-exonegene (forward)
• 100.0 10.0 (5'-ACA GAA ACT GAT ACT TAT-AT A GCG-3') mini-exonegene (reverse)
• 100.0 10.0 13A (5'-GTG GGG GAG GGG CGT TCT-3') kDNA (forward)
• 100.0 10.0 13B (5'-ATT TTC CAC CAA CCC CCA GTT-3') kDNA (Reverse)

The procedure of Polymerase Chain Reaction :

PCR reaction kit (PCR PreMix) was purchased from Bioneer company. The PCR reaction was carried out in 20 ?l solution containing (Taq DNA polymerase), 1 U, each dNTP (dATP, dGTP, dCTP, dTTP) 250 ?M, 1.5 mM MgCl2, Tris-HCl (pH 9.0), 10 mM, KCl, 30 mM, Template DNA, 5 - 50 ng, Primer, 5 - 10 pmole

The Reaction mixture was prepared in a pre-mix tube as shown in the following table(Table 3.2). according to BIONEER company instructions (Appendix No. 5).

After the addition was completed all the components of a mixed reaction in pre-mix tube mixed well by vortex, all tubes were centrifuged for 30 seconds at 10,000 rpm according to manufacture company, then all tubes were transferred into Thermal cycler apparatus for starting Thermal cycler program.