1. Introduction
There are many differing protocols and a large number of commercially available kits used for the extraction of DNA from whole blood. This procedure is one we use
routinely in both research and clinical service provision and is cheap and robust. It can also be applied to cell pellets from dispersed tissues or cell cultures (omitting the red blood lysis step.

2. Materials
This method uses standard chemicals that can be obtained from any major supplier;

we use Sigma.
1. Waterbath set at 65°C.
2. Centrifuge tubes (15 mL; Falcon).
3. Microfuge (1.5 mL) tubes.
4. Tube roller/rotator.
5. Glass Pasteur pipets, heated to seal the end and curled to form a “loop” or “hook” for spooling DNA.
6. EDTA (0.5 M), pH 8.0: Add 146.1 g of anhydrous EDTA to 800 mL of distilled water.
Adjust pH to 8.0 with NaOH pellets (this will require about 20 g). Make up to 1 L with distilled water. Autoclave at 15 p.s.i. for 15 min.
7. 1 M Tris-HCl, pH 7.6: Dissolve 121.1 g of Tris base in 800 mL of distilled water. Adjust pH with concentrated HCl (this requires about 60 mL). CAUTION: the addition of acid
produces heat. Allow mixture to cool to room temperature before finally correcting pH. Make up to 1 L with distilled water. Autoclave at 15 p.s.i. for 15 min.
8. Reagent A: Red blood cell lysis: 0.01M Tris-HCl pH 7.4, 320 mM sucrose, 5 mM MgCl2, 1% Triton X 100.
9. Add 10 mL of 1 M Tris, 109.54 g of sucrose, 0.47 g of MgCl2, and 10 mL of Triton X-100
to 800 mL of distilled water. Adjust pH to 8.0, and make up to 1 L with distilled water. Autoclave at 10 p.s.i. for 10 min .
10. Reagent B: Cell lysis: 0.4 M Tris-HCl, 150 mM NaCl, 0.06 M EDTA, 1% sodium dodecyl sulphate, pH 8.0. Take 400 mL of 1 M Tris (pH 7.6), 120 mL of 0.5 M EDTA (pH 8.0),
8.76 g of NaCl, and adjust pH to 8.0. Make up to 1 L with distilled water. Autoclave 15 min at 15. p.s.i. After autoclaving, add 10 g of sodium dodecyl sulphate.

11. 5 M sodium perchlorate: Dissolve 70 g of sodium perchlorate in 80 mL of distilled water. Make up to 100 mL.
12. TE Buffer, pH 7.6: Take 10 mL of 1 M Tris-HCl, pH 7.6, 2 mL of 0.5 M EDTA, and make up to 1 L with distilled water. Adjust pH to 7.6 and autoclave 15 min at 15. p.s.i.
13. Chloroform prechilled to 4°C.
14. Ethanol (100%) prechilled to 4°C.

3. Method

3.1. Blood Collection

1. Collect blood in either a heparin- or EDTA-containing Vacutainer by venipuncture Store at room temperature and extract within the same working day.

3.2. DNA Extraction
To extract DNA from cell cultures or disaggregated tissues, omit steps 1 through 3.
1. Place 3 mL of whole blood in a 15-mL falcon tube.
2. Add 12 mL of reagent A.
3. Mix on a rolling or rotating blood mixer for 4 min at room temperature.
4. Centrifuge at 3000g for 5 min at room temperature.
5. Discard supernatant without disturbing cell pellet. Remove remaining moisture by inverting
the tube and blotting onto tissue paper.
6. Add 1 mL of reagent B and vortex briefly to resuspend the cell pellet.
7. Add 250 μL of 5 M sodium perchlorate and mix by inverting tube several times.
8. Place tube in waterbath for 15 to 20 min at 65°C.
9. Allow to cool to room temperature.
10. Add 2 mL of ice-cold chloroform.
11. Mix on a rolling or rotating mixer for 30 to 60 min .

12. Centrifuge at 2400g for 2 min.
13. Transfer upper phase into a clean falcon tube using a sterile pipet.
14. Add 2 to 3 mL of ice-cold ethanol and invert gently to allow DNA to precipitate.

15. Using a freshly prepared flamed Pasteur pipet spool the DNA onto the hooked end .
16. Transfer to a 1.5-mL Eppendorf tube and allow to air dry .
17. Resuspend in 200 μL of TE buffer .

4. Notes
1. Autoclaving sugars at high temperature can cause caramelization (browning), which degrades the sugars.
2. As will all body fluids, blood represents a potential biohazard. Care should be taken in all steps requiring handling of blood. If the subject is from a known high risk category (e.g.,intravenous drug abusers) additional precautions may be required.
3. Rotation for less than 30 or over 60 min can reduce the DNA yield.
4. DNA should appear as a mucus-like strand in the solution phase.
5. Rotating the hooked end by rolling between thumb and forefinger usually works well. If the DNA adheres to the hook, break it off into the Eppendorf and resuspend the DNA before transferring to a fresh tube.
6. Ethanol will interfere with both measurements of DNA concentration and PCR reactions. However, overdrying the pellet will prolong the resuspension time.

7. The small amount of EDTA in TE will not affect PCR. We routinely use 1 μL per PCR reaction without adverse affects.
8. DNA can be quantified and diluted to a working concentration at this point or simply use 1 μL per PCR reaction; routinely, we expect 200 to 500 ng/μL DNA to be the yield
of this procedure.

Colorectal cancer, also called colon cancer or large bowel cancer, includes cancerous growths in the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is the fifth most common form of cancer in the United States and the third leading cause of cancer-related death in the Western world. Colorectal cancers arise from adenomatous polyps in the colon. These mushroom-shaped growths are usually benign, but some develop into cancer over time. Localized colon cancer is usually diagnosed through colonoscopy.

Signs and symptoms

• Feeling of incomplete defecation
• Change in bowel habit
• Reduction in size and shape of stool
• Black or brown stool (bleeding)
• Constipation, bowel pain, and vomiting may also be present.

Risk factors

• Age. The risk of developing colorectal cancer increases with age. Most cases occur in the 60s and 70s, while cases before age 50 are uncommon unless a family history of early colon cancer is present.
• Polyps of the colon, particularly adenomatous polyps, are a risk factor for colon cancer. The removal of colon polyps at the time of colonoscopy reduces the subsequent risk of colon cancer.
• History of cancer. Individuals who have previously been diagnosed and treated for colon cancer are at risk for developing colon cancer in the future. Women who have had cancer of the ovary, uterus, or breast are at higher risk of developing colorectal cancer.
• Heredity:
  
  • Family history of colon cancer, especially in a close relative before the age of 55 or multiple relatives.
  • Familial adenomatous polyposis (FAP) carries a near 100% risk of developing colorectal cancer by the age of 40 if untreated
  • Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome
• Smoking. Smokers are more likely to die of colorectal cancer than non-smokers. An American Cancer Society study found that "Women who smoked were more than 40% more likely to die from colorectal cancer than women who never had smoked. Male smokers had more than a 30% increase in risk of dying from the disease compared to men who never had smoked."
• Diet. Studies show that a diet high in red meat and low in fresh fruit, vegetables, poultry and fish increases the risk of colorectal cancer. In June 2005, a study by the European Prospective Investigation into Cancer and Nutrition suggested that diets high in red and processed meat, as well as those low in fiber, are associated with an increased risk of colorectal cancer. Individuals who frequently eat fish showed a decreased risk. However, other studies have cast doubt on the claim that diets high in fiber decrease the risk of colorectal cancer; rather, low-fiber diet was associated with other risk factors, leading to confounding. The nature of the relationship between dietary fiber and risk of colorectal cancer remains controversial.
• Physical inactivity. People who are physically active are at lower risk of developing colorectal cancer.
• Virus. Exposure to some viruses (such as particular strains of human papilloma virus) may be associated with colorectal cancer.
• Primary sclerosing cholangitis offers a risk independent to ulcerative colitis
• Low levels of selenium.
• Inflammatory bowel disease. About one percent of colorectal cancer patients have a history of chronic ulcerative colitis. The risk of developing colorectal cancer varies inversely with the age of onset of the colitis and directly with the extent of colonic involvement and the duration of active disease. Patients with colorectal Crohn's disease have a more than average risk of colorectal cancer, but less than that of patients with ulcerative colitis.
• Environmental factors. Industrialized countries are at a relatively increased risk compared to less developed countries that traditionally had high-fiber/low-fat diets. Studies of migrant populations have revealed a role for environmental factors, particularly dietary, in the etiology of colorectal cancers.
• Exogenous hormones. The differences in the time trends in colorectal cancer in males and females could be explained by cohort effects in exposure to some gender-specific risk factor; one possibility that has been suggested is exposure to estrogens.There is, however, little evidence of an influence of endogenous hormones on the risk of colorectal cancer. In contrast, there is evidence that exogenous estrogens such as hormone replacement therapy (HRT), tamoxifen, or oral contraceptives might be associated with colorectal tumors.
• Alcohol. Drinking, especially heavily, may be a risk factor.

Diagnosis

• Digital rectal exam (DRE): The doctor inserts a lubricated, gloved finger into the rectum to feel for abnormal areas. It only detects tumors large enough to be felt in the distal part of the rectum but is useful as an initial screening test.
• Fecal occult blood test (FOBT): a test for blood in the stool. Two types of tests can be used for detecting occult blood in stools i.e. guaiac based (chemical test) and immunochemical. The sensitivity of immunochemical testing is superior to that of chemical testing without an unacceptable reduction in specifity.
• Endoscopy:
  
  • Sigmoidoscopy: A lighted probe (sigmoidoscope) is inserted into the rectum and lower colon to check for polyps and other abnormalities.
  • Colonoscopy: A lighted probe called a colonoscope is inserted into the rectum and the entire colon to look for polyps and other abnormalities that may be caused by cancer. A colonoscopy has the advantage that if polyps are found during the procedure they can be immediately removed. Tissue can also be taken for biopsy.

• Double contrast barium enema (DCBE): First, an overnight preparation is taken to cleanse the colon. An enema containing barium sulfate is administered, then air is insufflated into the colon, distending it. The result is a thin layer of barium over the inner lining of the colon which is visible on X-ray films. A cancer or a precancerous polyp can be detected this way. This technique can miss the (less common) flat polyp.
• Virtual colonoscopy replaces X-ray films in the double contrast barium enema (above) with a special computed tomography scan and requires special workstation software in order for the radiologist to interpret. This technique is approaching colonoscopy in sensitivity for polyps. However, any polyps found must still be removed by standard colonoscopy.
• Standard computed axial tomography is an x-ray method that can be used to determine the degree of spread of cancer, but is not sensitive enough to use for screening. Some cancers are found in CAT scans performed for other reasons.
• Blood tests: Measurement of the patient's blood for elevated levels of certain proteins can give an indication of tumor load. In particular, high levels of carcinoembryonic antigen (CEA) in the blood can indicatemetastasis of adenocarcinoma. These tests are frequently false positive or false negative, and are not recommended for screening, it can be useful to assess disease recurrence.
• Genetic counseling and genetic testing for families who may have a hereditary form of colon cancer, such as hereditary nonpolyposis colorectal cancer (HNPCC) or familial adenomatous polyposis (FAP).
• Positron emission tomography (PET) is a 3-dimensional scanning technology where a radioactive sugar is injected into the patient, the sugar collects in tissues with high metabolic activity, and an image is formed by measuring the emission of radiation from the sugar. Because cancer cells often have very high metabolic rate, this can be used to differentiate benign and malignant tumors. PET is not used for screening and does not (yet) have a place in routine workup of colorectal cancer cases.
• Whole-Body PET imaging is the most accurate diagnostic test for detection of recurrent colorectal cancer, and is a cost-effective way to differentiate resectable from non-resectable disease. A PET scan is indicated whenever a major management decision depends upon accurate evaluation of tumour presence and extent.
• Stool DNA testing is an emerging technology in screening for colorectal cancer. Pre-malignant adenomas and cancers shed DNA markers from their cells which are not degraded during the digestive process and remain stable in the stool. Capture, followed by PCR amplifies the DNA to detectable levels for assay. Clinical studies have shown a cancer detection sensitivity of 71%–91%.

Prevention

• Colon cancer can be prevented by preliminary monitoring like bowel status after 50 years. You should check your bowel status by monitoring no. of stool per day and size and structure of stool. number of stool should not more than and not less than one times in 24 hr. Even you should monitor about size and shape, it should be neither too viscous nor too loose.
• Go for rectal digital examination. This will tell about formation of small nodules which is easier to remove and can controlled on primary status.
• You should also go for Occult blood examination in urine which tell us about any hidden blood loss or secretion of blood in gastrointestinal track.
• Take a good vitamin and high fiber diet which will improve your GIT healthiness.
• Regular physical exercise for 30 minute a day will be enough.

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