chapter 5 materials and methods

Materials and Methods 5

57 Dept. of pharmaceutical sciences,Saurashtra University,Rajkot.

CHAPTER 5 MATERIALS AND METHODS



5. MATERIALS AND METHODS

5.1 Specimen require

Blood sample collected in an EDTA vacutte is used because heparin is a

protein that creates hindrance in a PCR.

5.2 Reagent required

5.2.1 Reagents for DNA isolation.

5.2.1.1 TE: tris-Cl (pH 8) + EDTA (ethylene diamine tetra acetate) (Sigma c.

no.E5134)

5.2.1.2 RBC lysis buffer:

0.32M sucrose (to maintain isotonic environment). This solution

should filter prior to use.

10mM tris HCl(pH7.6) (maintain Ph)

5mM MgCl2 (maintain nuclear membrane integrity)

1% Triton 100 (Sigma c.no. t8787)

Make up to 1 L. do not autoclave

5.2.1.3 DNA extraction buffer:

1M tris buffer (Kemphasol, c. no. 2277)

HCl (Nice c. no. H11229)

0.5M EDTA (Sigma c. no.E5134) act as chelating agent (because

heavy metals promotes phosphodiester bond breakage)

10% SDS (anionic detergent, cell membrane is solubilize & thus

lyses cell, inhibit cellular DNase and denatured protein)

10% N-lauryl sarcosine (anionic detergent) (cat. No. L5125)

5.2.1.4 Proteinase-k (10mg/mL) (Sigma, p2380)

5.2.1.5 5M NaCl solution (Qualigen, c. no. 27605)

5.2.1.6 Alcohol (ethanol) (Baroda Chemical Ind.)



5.2.2 Reagents for RFLP-PCR

5.2.2.1 10 x Cetus buffer

2M KCl

1M Tris (pH 8.3) (Kemphasol, c. no. 2277)

1M MgCl2 (Ranbaxy, C. No. M0020)

Gelatin (300 blooms)

5.2.2.2 dNTPs (2.0 mMol)

5.2.2.3 Taq Polymerase (3.0 U/μL)

5.2.2.4 Forward Primer (Exon 7): (8 pM)

5’-AGACTATCAACTTAATTTCTGATCA-3’

5.2.2.5 Reverse Primer (Exon 7): (8 pM)

5’-CCTTCCTTCTTTTTGATTTTGTTT-3’

5.2.2.6 Forward Primer (Exon 8): (8 pM)

5’-GTAATAACCAAATGCAATGTGAA-3’

5.2.2.7 Reverse Primer (Exon 8): (8 pM)

5’-CTACAACACCCTTCTCACAG-3’

5.2.2.8 Genomic DNA

5.2.2.9 Distilled water

5.2.3 Reagents for restriction digestion

5.2.3.1 Dra I (10 U/ μL) (Bangalore GeNei)

5.2.3.2 Dde I (5 U/ μL) (Bangalore GeNei)



5.2.4 Reagents for Agarose gel electrophoresis

5.2.4.1 Agarose (Banglore GeNei, c. no. 105139)

5.2.4.2 EtBr

5.2.4.3 Bromophenol blue

5.2.4.4 Acetic Acid

5.2.4.5 Glycerol

5.3 Methodology

5.3.1 Principle

The molecular diagnosis of SMN gene deletions can be carried out by

polymerase chain reaction (PCR) followed by restriction fragment length

polymorphism (RFLP). The telomeric and centromeric copies in exon 7 and

exon 8 of SMN gene differ from each other by single base changes that can be

identified by selective restriction enzyme digestion of DNA. For exon 7, the

PCR amplified DNA product is digested with DraI restriction enzyme and

visualized with ethidium bromide on agarose gel electrophoresis. This allows

the SMN1 and SMN2 genes to be distinguished. Similarly for exon 8, the

restriction enzyme DdeI is used. In 5- 10% patients, deletion of both SMN1

and SMN2 genes are not detected. In such cases, point mutations have been

identified. However, detection of point mutation requires specialized

techniques like SSCP (single strand conformation polymorphism) and

heteroduplex analysis followed by DNA sequencing. Therefore, point mutation

analysis is not routinely performed for diagnostic purposes. Absence of exon 7

of SMN1 gene in peripheral blood has become a diagnostic tool for

confirmation of the disease. The test has a sensitivity of 95% and specificity is

over 99%. If SMA is diagnosed with strictly defined criteria a patient with exon

7 deletions has 99% chances of having SMA (Panigrahi et al. 2007).



5.3.2 Limitation of the method

In 5- 10% patients, deletion of both SMN1 and SMN2 genes are

not detected.

The restriction digestion method does not differentiate between a

true deletion and a gene conversion.

Gene conversion can be identified by SSCP.

Point mutation cannot be identified by this method (Scheffer et

al. 2001).

5.3.3 Hazards and precaution

Wear gloves during isolation of DNA to prevent contamination. EtBr

is a carcinogenic substance so take precaution while use. SDS is irritating

substance avoid inhalation. Use EDTA vacutte for collection of blood,

heparin decrease quality of DNA. Don’t insert pipettes in reagent bottle, to

avoid contamination. Maintain temperature during DNA extraction and

protein digestion to improve quality of the DNA. Store the enzymes in

mansion temperature to prevent degradation of them.

5.3.4 Isolation of DNA from blood

Take 3 ml of the sample blood in a centrifuge tube.

Add 2 to 3 volume of TE buffer.

Mix well and spin at 4000-5000 rpm for 15 minute.

Remove supernant.

Collect the pellet; add 2 to 3 volume of RBC lyses buffer.

Mix well and spin at 4000-5000 rpm for 15 minute.



Remove supernatant.

Repeat the process (2 to 3 times) to get white pellet.

Add 3mL DNA extraction buffer and Add 30µL proteinase k.

Incubate overnight at 37°C.

After overnight incubation pellet is completely dissolved.

Add 300µl 5M NaCl solution.

Add 6 ml chilled alcohol (ethanol).S

Spool out the DNA.

Wash the DNA pellet with 70% ethanol (2 to 3 times).

Leave in open air for drying.

Dissolved the dried DNA in required amount of 500 μL TE buffer.

Vortex it

Take 5 μL of DNA.

Add 995 μL of distilled water.

Determine the concentration by spectrophotometric method at λ max

260 and 280 nm. (Procedure optimized at Institute of Human Genetics,

satellite, Ahmedabad)



5.3.5 RFLP- PCR.

5.3.5.1 Preparation of reaction mixture

10X Cetus buffer 2.0 μL

Distilled water 13.2 μL

dNTPs (2 mMol) 2.0 μL

Primer F (8 pMol) 1.0 μL

Primer R (8 pMol) 1.0 μL

Taq DNA Polymerase 0.3 μL

Genomic DNA (500nG) ____

Take 2.0 μL of 10X Cetus buffer in a PCR tube.

Add 13.2 μL of distilled water to the tube.

Add 2.0 μL of dNTPs (2 mMol) to the PCR tube

Add 0.3 μL Taq DNA Polymerase to the mixture

Mix well the master mixture

To the master mixture add 1.0 μL of each Primer F (8 pMol) and Primer

R (8 pMol).

To the mixture add 500 nG of Genomic DNA

Mix well.

Put it for amplification as per following condition. (Procedure optimized

at Institute of Human Genetics, satellite, Ahmedabad)



5.3.5.2 Condition for amplification

Step 1 Initial Denaturation

94 oC for 5 minute 1 cycle

Step 2

Denaturation 94 oC 60sec

32 cycle Annealing 57 oC 60sec

Elongation 72 oC 60sec

Step 3 Final Extension

72 oC for 5 minute 1cycle

Table 4 PCR amplification condition

5.3.6 Restriction fragment length polymorphism (RFLP)

For exon 7, the PCR amplified DNA product is digested with DraI restriction

enzyme and for exon 8, the PCR amplified DNA product is digested with DedI

restriction enzyme.

Enzyme Enzyme Conc. (10 U/μL)

Product (μL)

Time for Digestion

Temp.

Dra I 1 μL (10 U/μL)

10 μL 24 hr 37˚C

Dde I 0.5 μL (5 U/μL)

10 μL 24 hr 37˚C

Table 5 Restriction digestion condition

To the 10 μL of the PCR product add 1 μL of Dra I (10 U/ μL) and 0.5 μL of

the Dde I (5 U/ μL) and keep it for digestion at 37˚C for 24 h. (Procedure

optimized at Institute of Human Genetics, satellite, Ahmedabad)



5.3.7 Agarose Gel Electrophoresis:

2.0 gm of agarose (GeNei-105139) dissolved in 100mL 1X TAE buffer.

Digest agarose in microwave oven till clear solution is obtained.

Add EtBr (1mg/ml) 15μL at 60 oC and mix gently.

Cast the gel in gel caster for 15 to 20 minute (till get it solidified).

Load the digested DNA (digested with Dra I and Dde I) + loading dye 1

μL (3X Bromophenol blue + Glycerol).

Run the gel electrophoresis at 150V until it run 1/3.

Observed under the UV trance illuminator.

5.4 Calculation

O.D. at 260nm =

O.D. at 280nm =

Concentration of DNA (nG/μL) = O.D. at 260 nm x 50 x 200 (dilution

factor).

A260/A280 should be in between 1.6 to 1.8

>1.8 Indicate impurity of protein.

<1.6 Indicate impurity of RNA.

(Procedure optimized at Institute of Human Genetics, satellite,

Ahmedabad)

chapter 5 materials and methods

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