Dna Was Extracted From Buccal Cells Biology Essay

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Buccal cells are collected from healthy persons, tobacco users tobacco chewing and smoking and oral cancer patients by using three types of mouthwashes like Listerine, 0.9%saline and Hexidine. The list of category of patients used different mouth washes are tabulated in the table 5. The DNA was extracted from buccal cells obtained from mouthwash samples and the stability is maintained by re-suspending in 0.5ml of TE buffer and stored.

Gel Electrophoresis

Agarose gel electrophoresis is performed for the identification of DNA from all the volunteers. It was shown that all the samples contained DNA was good and yielded enough amount of DNA.

In the figure 5 the DNA smears of the volunteer No P, A, I were seen in the lane 2, 5, 7 respectively that are already diagnosed with cancer, tobacco chewing and smoking where volunteer is healthy. In sample well 1, DNA marker (Z) was loaded.

Figure 6 showing the DNA smears of volunteer no B, L, M was a healthy and 2 cancer patient respectively and the lane is 4, 6, 8 and DNA marker at lane 2.

Figure 7 showed the agarose gel electrophoresis of healthy, cancer, and the person having the habits of tobacco chewing, smoking and alcohol drinking and their volunteer numbers were F, R, X. and lanes are 3, 5 and 8 respectively.DNA marker(Z) at lane 2.

Figure 8 showed the DNA smears of volunteer no H, Y, V who was diagnosed with healthy smoking with alcohol and tobacco chewing in the lane 2, 4, 6 and marker at lane 8.

Figure 9 shows the DNA smears of tobacco chewing with smoking and alcohol, cancer patient and tobacco chewer with smoking and their volunteer number is C, K, E, D at the lanes 3, 5, 6, 8 and DNA marker at lane 1.

Quantification of DNA

The yield of DNA from different volunteers who are tobacco users, smoking also healthy was found using UV spectrophotometer. The average yield of DNA was found to be 2.28µg. The results are shown in the table 6.

Comparison between mouthwash samples and their purity ratio

When compared between the mouthwashes used Listerine is found to have the high purity ratio and high yield. The results are shown in the table 7 and figure 10.

PCR study

The isolated DNA from different users yield was very good and more enough to amplify the genes. So among the 25 volunteers three volunteers (A, C, L) DNA were selected according to the yield and the gene is amplified and shown Figure 11.

Polymorphism study

The amplified gene was then subjected to study the polymorphism by restriction fragment length polymorphism. Restriction endonuclease (Hind III) was used to cut the genes in to fragments where every individual has unique restriction sites. And the volunteer (A, C, L) resulted in polymorphism. The figure 12 shows the polymorphism.

Table 5: Selection of Participants and Collection of Mouthwash Samples.

SL. No

V. No

AGE

GENDER

CATEGORY

MOUTHWASH

1

A

27

Male

Healthy

Listerine

2

B

20

Female

Healthy

Hexidine

3

C

29

Male

Tobacco chewer, Smoker and Alcohol

Listerine

4

D

41

Male

Smoker and Alcohol

Listerine

5

E

55

Male

Tobacco chewer and Smoker

0.9 % Saline

6

F

23

Male

Healthy

0.9 % Saline

7

G

58

Female

Tobacco chewer

Listerine

8

H

26

Male

Healthy

0.9 % Saline

9

I

27

Male

Tobacco chewer and Smoker

Hexidine

10

J

24

Male

Healthy

Hexidine

11

K

66

Male

Squamous cell carcinoma

0.9 % Saline

12

L

50

Male

Ca. Laryngo pharynx

Listerine

13

M

78

Male

Ca. Tongue

0.9 % Saline

14

N

70

Male

Ca. Tongue Anterior

0.9 % Saline

15

O

65

Female

Ca. Cricopharynx

Hexidine

16

P

70

Male

Ca. Hypopharynx

Listerine

17

Q

65

Male

Ca. Hypopharynx

Listerine

18

R

55

Female

Cervix

0.9 % Saline

19

S

59

Male

Ca. Tongue

Hexidine

20

T

70

Male

Ca. Epiglottis

0.9 % Saline

21

U

45

Male

Smoker and Alcohol

Hexidine

22

V

50

Female

Tobacco chewer

0.9 % Saline

23

W

38

Female

Tobacco chewer

0.9 % Saline

24

X

42

Male

Tobacco chewer, Smoker and Alcohol

Listerine

25

Y

29

Male

Smoker and Alcohol

Hexidine

Figure 5: Agarose Gel Electrophoresis

Figure 6: Agarose Gel Electrophoresis

Figure 7: Agarose Gel Electrophoresis

Figure 8: Agarose Gel Electrophoresis

D:\old all\old\HDU-PROJECT\agarose new version\4.jpg

Figure 9: Agarose Gel Electrophoresis

C:\Users\Hidayath\Desktop\agarose new version\5.jpg

Table 6: Quantification of DNA by using UV Spectrophotometer shows the amount of DNA extracted from different volunteer.

Vol. No

Absorbance at 260 nm

Absorbance at 280 nm

Absorbance ratio at 260/280

YIELD in µg

A

0.0574

0.0326

1.76

2.87

B

0.0397

0.0276

1.43

1.98

C

0.0560

0.0286

1.95

2.80

D

0.0511

0.0354

1.44

2.55

E

0.0524

0.0347

1.51

2.62

F

0.0413

0.0315

1.31

2.06

G

0.0524

0.0347

1.51

2.62

H

0.0512

0.0319

1.60

2.56

I

0.0439

0.0420

1.04

2.19

J

0.0461

0.0283

1.62

2.30

K

0.0465

0.0306

1.51

2.32

L

0.0513

0.0268

1.91

2.56

M

0.0461

0.0317

1.45

2.30

N

0.0478

0.0294

1.62

2.39

O

0.0391

0.0302

1.29

1.95

Contd….

Vol. No

Absorbance at 260 nm

Absorbance at 280 nm

Absorbance ratio at 260/280

YIELD in µg

P

0.0314

0.0263

1.93

1.57

Q

0.0391

0.0247

1.58

1.95

R

0.0399

0.0212

1.88

1.99

S

0.0515

0.0334

1.54

2.57

T

0.0498

0.0420

1.18

2.49

U

0.0377

0.0256

1.47

1.88

V

0.0446

0.0294

1.51

2.23

W

0.0368

0.0206

1.78

1.84

X

0.0457

0.0240

1.90

2.28

Y

0.0379

0.0229

1.65

1.89

Figure 10: Comparison between Mouthwashes

Table 7: Comparison of Average yield of DNA.

SL. No

Type of Mouthwash used

Average Yield of DNA (µg)

1

Listerine

2.3

2

0.9% Saline

2.25

3

Hexidine

1.8

Figure 11: Polymerase Chain Reaction

C:\Users\Hidayath\Desktop\Picture1.jpg

Lane-M Marker

Lane-1 Healthy Person (Volunteer-A)

Lane-2 Cancer Patient (Volunteer-L)

Lane-3 Tobacco Chewer (Volunteer-C)

Figure 12: Restriction Fragment Length Polymorphism

Lane-2 Marker

Lane-3 Healthy Person (Volunteer-A)

Lane-4 Tobacco Chewer (Volunteer-C)

Lane-5 Cancer Patient (Volunteer-L)

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