Introductory
These articles provide background information about the topics in DNA/Criminal Justice. They are selected for visitors who want a basic explanation of the concepts presented in this section.
Williams L. 2000, March 28. How DNA Analysis Works. The Gleaner. p. 8A.
Advanced
These articles provide expanded information about the topics in DNA and Criminal Justice. They are selected for visitors who want to explore more in-depth information about the concepts presented in this section.
Committee on DNA Forensic Science: An Update, National Research Council. 1996. The Evaluation of Forensic DNA Evidence. 272 pp.
Committee on DNA Technology in Forensic Science, National Research Council. 1992. DNA Technology in Forensic Science. 200 pp.
Kennedy D. 2003. Forensic Science: Oxymoron? Science 302 (5651): 1625.
Koshland DE. 1992. DNA Fingerprinting and Eyewitness Testimony. Science 256 (5057): 593.
Dec 8, 2007
DNA and Inherited Disease "hemochromatosis"
Inherited Disease Activity
A married couple...the Sheahans...introduce the scenario, regarding hemochromatosis and the risk factor for their three children. The presentation begins with a close-up of the father.
MICHAEL: Hundreds of diseases can be inherited through defective gene sequences. My six-year old daughter, Cassidy, suffers from an inherited genetic disorder called "hemochromatosis." This disease causes a person's intestines to absorb too much iron. The iron then builds up in different vital organs. Eventually, it can damage the liver, the heart, the joints...and possibly even cause death.
JENNIFER: My father, who is 54 became ill with hemochromatosis five years ago, and this was the first he knew that he had the disease. The symptoms...such as being overly tired and having varying degrees of pain...are shared with a lot of other diseases and they don't usually appear until middle age or later. When we discovered that there was also some incidence of the disease on Michael's side of the family, we decided to have Cassidy tested.
MICHAEL: Unfortunately, Cassidy's genetic tests showed that she has hereditary hemochromatosis. Now she goes weekly for a treatment known as a phlebotomy, in which they remove blood from her so that her body replenishes her own red blood cells. When she started, she was getting 40 cc's and now she's up to 75 cc's. We're hopeful that once she de-irons she can go on a maintenance program in which they won't have to take as much blood so often.
JENNIFER: Even though she's still young, she's aware that she has the disease. She's very resilient and hasn't shown any ill effects. We try to help her by doing such things as cooking without iron skillets or much red meat. It's ongoing maintenance.
MICHAEL: We have two younger children, Callie and Michael, who could also be at risk, since Jennifer and I are both carriers even though we don't have the disease. So we felt that they had to be tested, too.
NARRATOR : In cases such as the Sheahans, comparing the gene sequences from a normal gene to the child's DNA will provide the test results. If one or both of the child's genes...from the mother and the father...is normal, then the child will be okay or, at the worst, a carrier of the disorder. But if both genes have a hemochromatosis defect, the child could develop hemochromatosis.
One pair of signs comes to life. The right half of each sign fills with letters, but they rapidly advance through the gene sequence and stop at roughly 30 letters away from the mutation, which is located 285 letters into the gene.
NARRATOR: You can see that the top row of letters is the gene sequence from the child's DNA. The lower row is what we hope to see: the normal gene sequence.
Use the control buttons to scroll through the gene and hunt for a difference between the two lines of letters. Any difference will be a mutation. This particular gene is 2,727 letters long, and you can watch your progress in the small window on the left side of the screen.
If you think you've found a mutation, press the red button.
(Visitors can scroll forward and backward, fast or slow, until they find the mutation and press the button. The most common mutation is a single letter change around position 285...we will verify. A pop-up window responds to wrong answers with :
NARRATOR (v.o.):
You haven't found a mutation - keep trying or touch 'find it for me'
A correct answer activates the next video segment.
NARRATOR: You found a mutation, the most common of the two possible mutations. It is involved in 88% all hemochromatosis cases. So we already know that the child is at least a carrier.
(The second pair of signs comes to life and holds at the red reference line.)
NARRATOR: The Centers for Disease Control says that people with just one copy of the defective gene rarely have excess iron build-up. So the child will probably be okay if the other copy is normal. Now let's see what the second sequence looks like.
(The activity is identical to the first try, but there are three possible outcomes this time:
{1} no mutation, {2} the same mutation seen in the first gene, and {3} the second most common mutation. The program searches the first mutation site, then advances rapidly to the site of the second most-common mutation. The visitor decides whether to keep searching the first site or to advance to the second, based on an on-screen prompt saying that he/she has passed over the mutation region. The exhibit is programmed to offer one of these three outcomes randomly to each visitor. Each outcome includes a different video sequence ending, but all contain the same basic information.
Outcome 1
NARRATOR: You didn't find a mutation. The second gene is normal. Though the child shouldn't suffer from iron overload, the earlier defective gene means the child could pass the defect along to any of his or her own children.
JENNIFER: Thousands of diseases are related to defective gene sequences. You can inherit defective mutations from one or both of your parents, and you can pass them along to your children, increasing their risk, too. But inheriting a genetic defect doesn't necessarily mean you'll get sick.
MICHAEL: At first, doctors didn't want to test Cassidy because they said it was a middle-age disease. Though there is no cure for hemochromatosis at present, by discovering it early as we did, we can possibly negate much of the damage before it occurs.
JENNIFER: We did, in fact, have our other two children tested. Each had only one defective gene, meaning that they are carriers but unlikely to develop hemochromatosis themselves. In the meantime, we're optimistic for Cassidy and we'll help her to live as normal a life as possible.
Outcome 2
NARRATOR: You found a second mutation, which means that the child is at risk for developing the disease too. In this case, it too is the most common version of the mutation. So what can be done? Please read the CDC's recommendations next to this screen.
CDC Recommendations:
Using DNA To Maintain Good Health
If a child is known to have inherited the genetic mutations for hemochromatosis, the impact of the disease can often be reduced. Symptoms of the disease usually do not appear until middle age. Even in such cases, health and life expectancy can be improved through a treatment known as "phlebotomy," in which iron-rich blood is removed from the patient every week and replenished with normal blood by the body.
The Centers for Disease Control (CDC) recommends avoiding vitamins that contain iron and restricting vitamin C, which increases iron absorption. The CDC also recommends avoiding behavior that could damage the liver, such as more than mild alcohol consumption. Although patients may eat iron-containing foods, they should avoid eating raw seafood and shellfish, because iron-overload patients are susceptible to infections that these foods may carry.
JENNIFER: Thousands of diseases are related to defective gene sequences. You can inherit defective mutations from one or both of your parents, and you can pass them along to your children, increasing their risk, too. But inheriting a genetic defect doesn't necessarily mean you'll get sick.
MICHAEL: At first, doctors didn't want to test Cassidy because they said it was a middle-age disease. Though there is no cure for hemochromatosis at present, by discovering it early as we did, you can negate much of the damage before it occurs.
JENNIFER: We did, in fact, have our other two children tested. Each had only one defective gene, meaning that they are carriers but unlikely to develop hemochromatosis themselves. In the meantime, we're optimistic for Cassidy and we'll help her to live as normal a life as possible.
Outcome 3
NARRATOR (v.o.): You found a second mutation. In this case, it is a different mutation than was found in the first gene. The second mutation is less common but can also cause hemochromatosis, which means that the child is at risk for developing the disease too. So what can be done? Please read the CDC's recommendations above.
JENNIFER: Thousands of diseases are related to defective gene sequences. You can inherit defective mutations from one or both of your parents, and you can pass them along to your children, increasing their risk, too. But inheriting a genetic defect doesn't necessarily mean you'll get sick.
MICHAEL: At first, doctors didn't want to test Cassidy because they said it was a middle-age disease. Though there is no cure for hemochromatosis at present, by discovering it early as we did, you can negate much of the damage before it occurs.
JENNIFER: We did, in fact, have our other two children tested. Each had only one defective gene, meaning that they are carriers but unlikely to develop hemochromatosis themselves. In the meantime, we're optimistic for Cassidy and we'll help her to live as normal a life as possible.
End
A married couple...the Sheahans...introduce the scenario, regarding hemochromatosis and the risk factor for their three children. The presentation begins with a close-up of the father.
MICHAEL: Hundreds of diseases can be inherited through defective gene sequences. My six-year old daughter, Cassidy, suffers from an inherited genetic disorder called "hemochromatosis." This disease causes a person's intestines to absorb too much iron. The iron then builds up in different vital organs. Eventually, it can damage the liver, the heart, the joints...and possibly even cause death.
JENNIFER: My father, who is 54 became ill with hemochromatosis five years ago, and this was the first he knew that he had the disease. The symptoms...such as being overly tired and having varying degrees of pain...are shared with a lot of other diseases and they don't usually appear until middle age or later. When we discovered that there was also some incidence of the disease on Michael's side of the family, we decided to have Cassidy tested.
MICHAEL: Unfortunately, Cassidy's genetic tests showed that she has hereditary hemochromatosis. Now she goes weekly for a treatment known as a phlebotomy, in which they remove blood from her so that her body replenishes her own red blood cells. When she started, she was getting 40 cc's and now she's up to 75 cc's. We're hopeful that once she de-irons she can go on a maintenance program in which they won't have to take as much blood so often.
JENNIFER: Even though she's still young, she's aware that she has the disease. She's very resilient and hasn't shown any ill effects. We try to help her by doing such things as cooking without iron skillets or much red meat. It's ongoing maintenance.
MICHAEL: We have two younger children, Callie and Michael, who could also be at risk, since Jennifer and I are both carriers even though we don't have the disease. So we felt that they had to be tested, too.
NARRATOR : In cases such as the Sheahans, comparing the gene sequences from a normal gene to the child's DNA will provide the test results. If one or both of the child's genes...from the mother and the father...is normal, then the child will be okay or, at the worst, a carrier of the disorder. But if both genes have a hemochromatosis defect, the child could develop hemochromatosis.
One pair of signs comes to life. The right half of each sign fills with letters, but they rapidly advance through the gene sequence and stop at roughly 30 letters away from the mutation, which is located 285 letters into the gene.
NARRATOR: You can see that the top row of letters is the gene sequence from the child's DNA. The lower row is what we hope to see: the normal gene sequence.
Use the control buttons to scroll through the gene and hunt for a difference between the two lines of letters. Any difference will be a mutation. This particular gene is 2,727 letters long, and you can watch your progress in the small window on the left side of the screen.
If you think you've found a mutation, press the red button.
(Visitors can scroll forward and backward, fast or slow, until they find the mutation and press the button. The most common mutation is a single letter change around position 285...we will verify. A pop-up window responds to wrong answers with :
NARRATOR (v.o.):
You haven't found a mutation - keep trying or touch 'find it for me'
A correct answer activates the next video segment.
NARRATOR: You found a mutation, the most common of the two possible mutations. It is involved in 88% all hemochromatosis cases. So we already know that the child is at least a carrier.
(The second pair of signs comes to life and holds at the red reference line.)
NARRATOR: The Centers for Disease Control says that people with just one copy of the defective gene rarely have excess iron build-up. So the child will probably be okay if the other copy is normal. Now let's see what the second sequence looks like.
(The activity is identical to the first try, but there are three possible outcomes this time:
{1} no mutation, {2} the same mutation seen in the first gene, and {3} the second most common mutation. The program searches the first mutation site, then advances rapidly to the site of the second most-common mutation. The visitor decides whether to keep searching the first site or to advance to the second, based on an on-screen prompt saying that he/she has passed over the mutation region. The exhibit is programmed to offer one of these three outcomes randomly to each visitor. Each outcome includes a different video sequence ending, but all contain the same basic information.
Outcome 1
NARRATOR: You didn't find a mutation. The second gene is normal. Though the child shouldn't suffer from iron overload, the earlier defective gene means the child could pass the defect along to any of his or her own children.
JENNIFER: Thousands of diseases are related to defective gene sequences. You can inherit defective mutations from one or both of your parents, and you can pass them along to your children, increasing their risk, too. But inheriting a genetic defect doesn't necessarily mean you'll get sick.
MICHAEL: At first, doctors didn't want to test Cassidy because they said it was a middle-age disease. Though there is no cure for hemochromatosis at present, by discovering it early as we did, we can possibly negate much of the damage before it occurs.
JENNIFER: We did, in fact, have our other two children tested. Each had only one defective gene, meaning that they are carriers but unlikely to develop hemochromatosis themselves. In the meantime, we're optimistic for Cassidy and we'll help her to live as normal a life as possible.
Outcome 2
NARRATOR: You found a second mutation, which means that the child is at risk for developing the disease too. In this case, it too is the most common version of the mutation. So what can be done? Please read the CDC's recommendations next to this screen.
CDC Recommendations:
Using DNA To Maintain Good Health
If a child is known to have inherited the genetic mutations for hemochromatosis, the impact of the disease can often be reduced. Symptoms of the disease usually do not appear until middle age. Even in such cases, health and life expectancy can be improved through a treatment known as "phlebotomy," in which iron-rich blood is removed from the patient every week and replenished with normal blood by the body.
The Centers for Disease Control (CDC) recommends avoiding vitamins that contain iron and restricting vitamin C, which increases iron absorption. The CDC also recommends avoiding behavior that could damage the liver, such as more than mild alcohol consumption. Although patients may eat iron-containing foods, they should avoid eating raw seafood and shellfish, because iron-overload patients are susceptible to infections that these foods may carry.
JENNIFER: Thousands of diseases are related to defective gene sequences. You can inherit defective mutations from one or both of your parents, and you can pass them along to your children, increasing their risk, too. But inheriting a genetic defect doesn't necessarily mean you'll get sick.
MICHAEL: At first, doctors didn't want to test Cassidy because they said it was a middle-age disease. Though there is no cure for hemochromatosis at present, by discovering it early as we did, you can negate much of the damage before it occurs.
JENNIFER: We did, in fact, have our other two children tested. Each had only one defective gene, meaning that they are carriers but unlikely to develop hemochromatosis themselves. In the meantime, we're optimistic for Cassidy and we'll help her to live as normal a life as possible.
Outcome 3
NARRATOR (v.o.): You found a second mutation. In this case, it is a different mutation than was found in the first gene. The second mutation is less common but can also cause hemochromatosis, which means that the child is at risk for developing the disease too. So what can be done? Please read the CDC's recommendations above.
JENNIFER: Thousands of diseases are related to defective gene sequences. You can inherit defective mutations from one or both of your parents, and you can pass them along to your children, increasing their risk, too. But inheriting a genetic defect doesn't necessarily mean you'll get sick.
MICHAEL: At first, doctors didn't want to test Cassidy because they said it was a middle-age disease. Though there is no cure for hemochromatosis at present, by discovering it early as we did, you can negate much of the damage before it occurs.
JENNIFER: We did, in fact, have our other two children tested. Each had only one defective gene, meaning that they are carriers but unlikely to develop hemochromatosis themselves. In the meantime, we're optimistic for Cassidy and we'll help her to live as normal a life as possible.
End
Dec 6, 2007
Gene (DNA) Transfection Resources
Gene Transfection
1.Basic knowledge of gene transfection.
2.Protocol 1. Gene transfection and its related issues. (A must read manual).
3.Protocol 2. Cationic polymer transfection method.
4.Protocol 3. Calcium phosphate precipitation
5.Protocol 4. Calcium-phosphate-mediated transfection
6.Protocol 5. Lipofectamine transfection protocol
7.Protocol 6. Work with retrovirus vector.
8.Protocol 7. Work with adenovirus.
1.Basic knowledge of gene transfection.
2.Protocol 1. Gene transfection and its related issues. (A must read manual).
3.Protocol 2. Cationic polymer transfection method.
4.Protocol 3. Calcium phosphate precipitation
5.Protocol 4. Calcium-phosphate-mediated transfection
6.Protocol 5. Lipofectamine transfection protocol
7.Protocol 6. Work with retrovirus vector.
8.Protocol 7. Work with adenovirus.
Gene (DNA) Knochout Technology
Gene knockout=2007 Nobel Prize in Medicine knockin
(VOA Special English Health Report)
This year's Nobel Prize in medicine will go to three researchers who found a way to learn about the duties of individual genes. They discovered how to inactivate, or knock out, single genes in laboratory animals. The result is known as "knockout mice." The Karolinska Institute named the winners last week. Two Americans, Mario Capecchi and Oliver Smithies, will share the one and one-half million dollar prize with Martin Evans of Britain. They will receive what is officially called the Nobel Prize in Physiology or Medicine at a ceremony in Stockholm, Sweden, on December tenth.In the nineteen eighties, Mario Capecchi and Oliver Smithies both studied cells in mice to find how to target individual genes for changes. But the kinds of cells they independently studied could not be used to create gene-targeted animals. Martin Evans had the solution. He developed embryonic stem cells that could produce mice that carried new genetic material.The research greatly expanded knowledge about embryonic development as well as aging and disease. It led to a new technology -- gene targeting. And this has already produced five hundred mouse models of human conditions. Knockout mice are used for general research and for the development of new treatments. International efforts aim to make them available in the near future for all genes. Mario Capecchi is a researcher at the University of Utah. He was born in Italy in nineteen thirty-seven. He was homeless and on his own for years as a young boy. His mother had been sent to a Nazi German death camp. But she survived, and after she was freed she found him in a hospital. He was nine years old and being treated for severe malnutrition. They came to the United States where he entered school for the first time. Later, he became an American citizen. Oliver Smithies was born in Britain in nineteen twenty-five and also became an American citizen. He is a professor at the University of North Carolina. And, at age fifty, he learned to fly. He flies a motor glider and small airplanes. Martin Evans was born in nineteen forty-one, also in Britain. He is director of the School of Biosciences at Cardiff University inWales. He called winning the Nobel Prize "a boyhood dream come true."
Gene knockout protocols
Protocol 1. Gene knockout for generating delete mutations
2.Elegant gene knockout protocols
3.Gene knockout (mutagenesis and harvest)
4. Gene knockout in murine embryonic stem (ES) cells
(VOA Special English Health Report)
This year's Nobel Prize in medicine will go to three researchers who found a way to learn about the duties of individual genes. They discovered how to inactivate, or knock out, single genes in laboratory animals. The result is known as "knockout mice." The Karolinska Institute named the winners last week. Two Americans, Mario Capecchi and Oliver Smithies, will share the one and one-half million dollar prize with Martin Evans of Britain. They will receive what is officially called the Nobel Prize in Physiology or Medicine at a ceremony in Stockholm, Sweden, on December tenth.In the nineteen eighties, Mario Capecchi and Oliver Smithies both studied cells in mice to find how to target individual genes for changes. But the kinds of cells they independently studied could not be used to create gene-targeted animals. Martin Evans had the solution. He developed embryonic stem cells that could produce mice that carried new genetic material.The research greatly expanded knowledge about embryonic development as well as aging and disease. It led to a new technology -- gene targeting. And this has already produced five hundred mouse models of human conditions. Knockout mice are used for general research and for the development of new treatments. International efforts aim to make them available in the near future for all genes. Mario Capecchi is a researcher at the University of Utah. He was born in Italy in nineteen thirty-seven. He was homeless and on his own for years as a young boy. His mother had been sent to a Nazi German death camp. But she survived, and after she was freed she found him in a hospital. He was nine years old and being treated for severe malnutrition. They came to the United States where he entered school for the first time. Later, he became an American citizen. Oliver Smithies was born in Britain in nineteen twenty-five and also became an American citizen. He is a professor at the University of North Carolina. And, at age fifty, he learned to fly. He flies a motor glider and small airplanes. Martin Evans was born in nineteen forty-one, also in Britain. He is director of the School of Biosciences at Cardiff University inWales. He called winning the Nobel Prize "a boyhood dream come true."
Gene knockout protocols
Protocol 1. Gene knockout for generating delete mutations
2.Elegant gene knockout protocols
3.Gene knockout (mutagenesis and harvest)
4. Gene knockout in murine embryonic stem (ES) cells
DNA Southern Blot Protocol
Southern Blot Hybridization Protocols
Protocol 2. Protocol for Southern Blotting of Genimic DNA
Protocol 3. DNA Analysis by Southern Blotting
Protocol 4. Southern Blot Analysis
Protocol 4. Southern Blot Method
Commonly Used Reagents and Buffer Solutions for Southern Blot
3M ammonium acetate
Dissolve 231.24g of ammonium acetate in 1 liter dH2O.
10X TBE
100 gm/L (0.89M Tris) MW 121.14
55 gm/L (0.89M Boric acid) MW 61.83
40ml of 0.5M EDTA disodium
Adjust pH to 8.0 with 1:10 diluted HCl.
20X SSC
3M NaCl
0.3M sodium citrate
100X Denhardt's Solution
2% bovine serum albumin
2% polyvinylpyrrolidone [Sigma]
2% ficoll
Denaturing Solution
0.5M NaOH
1.0M NaCl
DNA Loading Dye
15% ficoll [F-4375 Sigma]
10% glycerol
0.05% bromophenol blue
1X TBE buffer
Weigh 15g of ficoll and add 50-100mg of bromophenol blue, then add 10ml of glycerol and 90ml of 1X TBE.
Place this mixture on a hot plate with a magnetic stirrer and allow to dissolve and make up to 100ml with 1X TBE.
DNA Lysis Buffer
100mM NaCl
10mM Tris-HCl pH 8.0
25mM EDTA (disodium salt)
0.5% of sodium dodecyl sulphate
0.1mg/ml Proteinase K
Ethidium Bromide
10mg of ethidium bromide powder
1ml of dH2O
Neutralizing Solution
1.5M Tris (pH 8.0)
1.0M NaCl
Protocol 2. Protocol for Southern Blotting of Genimic DNA
Protocol 3. DNA Analysis by Southern Blotting
Protocol 4. Southern Blot Analysis
Protocol 4. Southern Blot Method
Commonly Used Reagents and Buffer Solutions for Southern Blot
3M ammonium acetate
Dissolve 231.24g of ammonium acetate in 1 liter dH2O.
10X TBE
100 gm/L (0.89M Tris) MW 121.14
55 gm/L (0.89M Boric acid) MW 61.83
40ml of 0.5M EDTA disodium
Adjust pH to 8.0 with 1:10 diluted HCl.
20X SSC
3M NaCl
0.3M sodium citrate
100X Denhardt's Solution
2% bovine serum albumin
2% polyvinylpyrrolidone [Sigma]
2% ficoll
Denaturing Solution
0.5M NaOH
1.0M NaCl
DNA Loading Dye
15% ficoll [F-4375 Sigma]
10% glycerol
0.05% bromophenol blue
1X TBE buffer
Weigh 15g of ficoll and add 50-100mg of bromophenol blue, then add 10ml of glycerol and 90ml of 1X TBE.
Place this mixture on a hot plate with a magnetic stirrer and allow to dissolve and make up to 100ml with 1X TBE.
DNA Lysis Buffer
100mM NaCl
10mM Tris-HCl pH 8.0
25mM EDTA (disodium salt)
0.5% of sodium dodecyl sulphate
0.1mg/ml Proteinase K
Ethidium Bromide
10mg of ethidium bromide powder
1ml of dH2O
Neutralizing Solution
1.5M Tris (pH 8.0)
1.0M NaCl
SSCP Detect DNA Mutation
DNA Mutation Detection by SSCP
Single-Strand Conformational Polymorphism (SSCP)
PCR Protocol
10X PCR Buffer 1.0ul
dNTPs mix 1.0ul
10 mM Primer-F (ug/ul) 1.0ul
10 mM Primer-R (ug/ul) 1.0ul
Taq (2U) 1.0ul
32P dCTP (10uci/ul) 0.1ul
ddH2O 2.9ul
SUBTOTAL 8.0ul
Template DNA (50-100ng) 2.0ul
# dNTPS mix (final concentration): dATP 2.5mM; dTTP 2.5mM; dGTP 2.5mM; dCTP 1.25mM
Run the PCR with standard procedure, however, it should be optimized accordingly.
SSCP Gels
Prepare 0.5x MDE gel as follows:
MDE gel 16
ddH2O 44.2ml
10X TBE 3.84ml
10% APS 256ul
TEMED 25.6ul
Pour sequencing gel format with appropriate comb. Gel will polymerize in about 1 hour.
Loading Buffer
95% formamide
10mM NaOH
0.025% Bromophenol Blue
0.025% Xylene Cyanol
Run gel in 1X TEB buffer.
Heat denature samples at 94¡ãC for 5 minutes and place them on ice for 3-5 minutes. Load 2.0-4.0¦Ìl per sample. Include non-denatured controls.
Electrophoresis conditions
Fragment Size: 150-200 bp
6 Watts
10-12 hours
room temperature
Fragment Size: > 200 bp
8 Watts
10-12 hours
room temperature
Autoradiography
Dry gel and expose either at room temperature for 2 hours or at 180 centigrade for 16-18 hours.
Single-Strand Conformational Polymorphism (SSCP)
PCR Protocol
10X PCR Buffer 1.0ul
dNTPs mix 1.0ul
10 mM Primer-F (ug/ul) 1.0ul
10 mM Primer-R (ug/ul) 1.0ul
Taq (2U) 1.0ul
32P dCTP (10uci/ul) 0.1ul
ddH2O 2.9ul
SUBTOTAL 8.0ul
Template DNA (50-100ng) 2.0ul
# dNTPS mix (final concentration): dATP 2.5mM; dTTP 2.5mM; dGTP 2.5mM; dCTP 1.25mM
Run the PCR with standard procedure, however, it should be optimized accordingly.
SSCP Gels
Prepare 0.5x MDE gel as follows:
MDE gel 16
ddH2O 44.2ml
10X TBE 3.84ml
10% APS 256ul
TEMED 25.6ul
Pour sequencing gel format with appropriate comb. Gel will polymerize in about 1 hour.
Loading Buffer
95% formamide
10mM NaOH
0.025% Bromophenol Blue
0.025% Xylene Cyanol
Run gel in 1X TEB buffer.
Heat denature samples at 94¡ãC for 5 minutes and place them on ice for 3-5 minutes. Load 2.0-4.0¦Ìl per sample. Include non-denatured controls.
Electrophoresis conditions
Fragment Size: 150-200 bp
6 Watts
10-12 hours
room temperature
Fragment Size: > 200 bp
8 Watts
10-12 hours
room temperature
Autoradiography
Dry gel and expose either at room temperature for 2 hours or at 180 centigrade for 16-18 hours.
DNA Extraction from Agarose or Acrylamide Gel
DNA Extraction from Agarose or Acrylamide Gel
Protocol 1: DNA Fragment Purification from Agarose or Acrylamide
Protocol 2: Electro-elution of nucleic acids from agarose and polyacrylamide
Protocol 1: DNA Fragment Purification from Agarose or Acrylamide
Protocol 2: Electro-elution of nucleic acids from agarose and polyacrylamide
Deoxyribose Isolation from DNA Degrasion
Degrade DNA to Get Deoxyribose for GC/MS Analysis
1. Dissolve 100ug DNA in 200ul distilled water.
2. Add MgCL2 and sodium acetate to the final concentration of 15mM and 10mM, respectively.
3. Incubate with 2 units DNAse I at room temperature for 15 minutes.
4. Add ammonium bicarbonate to the final concentration of 100mM, and then incubate with 2mU phosphodiesterase at 37oC in water bath for 2 hours.
5. Add 1 unit of alkaline phosphatase and incubated at 37oC water bath for another 1 hour.
6. Air dry the solution with degraded DNA. Derivalize produced deoxyribose to its aldonitrile
acetate form as usual for analysis on GC/MS spectrometer.
1. Dissolve 100ug DNA in 200ul distilled water.
2. Add MgCL2 and sodium acetate to the final concentration of 15mM and 10mM, respectively.
3. Incubate with 2 units DNAse I at room temperature for 15 minutes.
4. Add ammonium bicarbonate to the final concentration of 100mM, and then incubate with 2mU phosphodiesterase at 37oC in water bath for 2 hours.
5. Add 1 unit of alkaline phosphatase and incubated at 37oC water bath for another 1 hour.
6. Air dry the solution with degraded DNA. Derivalize produced deoxyribose to its aldonitrile
acetate form as usual for analysis on GC/MS spectrometer.
DNA Precipitation and Purification
1.Prepare or obtain Buffered phenol,pH 8.Add 2 crystals of 8-Hydroxyquinoline to prevent oxidation.This also identifies the organic phase as yellow-colored.
2.Combine DNA sample with an equal volume of Buffered Phenol.
3.Mix well by vortexing until uniformly milky-yellow.
4.Centrifuge for 10 min at 4℃.
5.Promptly remove upper aqueous phase and transfer into a new tube.DO NOT remove the interface.
6.Add an equal volume of 24:1℃hloroform:Isoamyl alcohol.
7.Mix well by shaking the tubes.Vortexing does not help.
8.Centrifuge for 3 min at 4℃.
9.Remove supernatant and transfer to a new tube.
10.For small amounts of DNA,add 2 ml 1% linear Polyacrylamide carrier for each 100 ml of sample.
11.Mix well.Add 2.5 volumes of 95% ethanol.Mix by inversion.
12.For small amounts of DNA,incubate at -20℃ for 30 min.Centrifuge for 15 min.For large quantities of DNA (e.g.chromosomal preps),centrifuge immediately for 3 min.
13.Remove supernatant.Add a large volume of 70% ethanol.Mix well to dislodge the pellet.
14.Centrifuge for 2 min.Remove the supernatant and air dry.
15.Resuspend DNA in water or TE.
DNA Extraction from Paraffin Embedded Tissue
This is a four day procedure so it's best to start on Monday or Tuesday.
CASE SELECTION:
H&E stained thin sections are first reviewed by a pathologist, and areas of interest are outlined. Tissue blocks are then cut as follows:
3 consecutive 5-micron sections from a formalin fixed paraffin embedded block are cut and placed onto positively charged slides (some dissection protocols discourage use of charged slides). More can be cut if additional studies (e.g. immunohistochemistry) are being considered.
Stain the middle slide with H&E. Check that areas from the previous H&E slide are the same and if needed get the pathologist to review the new H&E slide (some small lesions will change diagnosis or even disappear in deeper sections). If the two H&E's look the same then Stain one or both with (see protocol below).
If the histopathology is at all difficult, photos are taken of areas to be dissected on both the methyl green and H&E slides. In general, the methyl green slide is photographed at a higher power than the H&E.
The pathologist then reviews the methyl green photo along side of the H&E slide. The methyl green photo is then marked by the pathologist to define the areas to be microdissected. If additional areas with no photos are suggested for use, mark the H&E slide, then take photos while dissecting to at least document what is taken. If there is any uncertainty, ask the Pathologist to re-review the area on the slide in question.
Day 1:
DISSECTION:
See photos of , , and .
Label the top of 0.5 ml pcr tubes, color code dot label them also.
Add 15 μl of 1X Buffer into each tube, close caps.
Do one case at a time. If normal is being dissected do this first.
Scrape away areas that you dont want away from the sample (use a #11 blade).
Take a new #15 blade and dip (don't dunk) tip of blade into the labeled tube with pcr pk buffer, put this very small drop from the tip of blade onto the sample area by tapping.
Start scraping tissue into the center of puddle. Watch puddle dry up to point where you can pick up the sample scrapings (do not overdry or tissue will fly away!!).
Place this carefully into the 0.5 ml pcr tube with the pcr/pk buffer.
The amount of pcr/pk buffer depends on the sample. In general it's 15 μl for anything less than 1 mm, and proportionally more if larger.
Go onto next area. Remember to use diiferent blades for each part of the tumor.
Overlay with 20 μl mineral oil. Close cap and seal with parafilm.
DIGESTION:
Incubate overnight for shaking at 120 rpm, 55℃.
Day 2:
Add 0.3 μl fresh conc (20 mg/ml stock) through the oil to sample. (It's 0.3 μl per 15ul original volume, so adjust as necessary).
Incubate overnight at 55℃.
Day 3:
(Repeat:) Add 0.3 μl fresh conc (20 mg/ml stock) through the oil to sample. (It's 0.3 μl per 15 μl original volume, so adjust as necessary).
Incubate overnight at 55℃.
Day 4:
Remove all parafilm from tubes. Inactivate Proteinase K for 10-15 minutes at 95 ℃ in PCR machine or hot water bath.
Remove oil by rolling samples and oil on parafilm and and pipette aqueous DNA into new tube.
SOLUTIONS
Digestion buffer: 1X PCR Buffer with 0.5% Tween20 / 0.4mg/ml pk stored in aliquots at -20 ℃.
PK: Stored as 20mg/ml aliquots at -20 ℃; can be refrozen a few times.
Methyl Green Staining
Methyl Green is used for staining of microdissected slides since we have found that hematoxylin (H&E) can interfere with PCR amplification. It is usually only necessary to stain and microdissect one slide, but two slides are used when the target is less than 1 mm in diameter.
A. Deparaffinization:
Xylene 2X 3 mins each.
100% ethanol 2 mins.
95% ethanol 2 mins.
70% ethanol 2 mins.
50% ethanol 2 mins.
dH2O 2 mins.
B. Staining:
0.1-1% Methyl green (dip 3-6 times or leave ~15-60 seconds).
The section should not be too dark. The methyl green concentration can be varied if sections are generally too dark or too light. It's better to start with a lower concentration.
dH2O 1-5 mins (depending on how blue/green the tissue is).
Air dry upright.
The slide is now ready for photographing and microdissection. It is better to use soon but can be stored for a few weeks if necessary.
Isolation DNA,RNA and Protein from same samples.
Isolation Genomic DNA,RNA,and Protein simultanously from same tissue or cells
Protocol 1. Isolation of DNA,RNA,and Protein simultaneously.
Protocol 2. Isolation of DNA,RNA,and Protein simultaneously (AllPrep DNA/RNA/Protein).
Protocol 3. Flow chart of isolation DNA,RNA,and Protein from same biomaterials.
Detailed procedure (click here)
Reference 1.Riol H, et al.Optimized Lymphocyte Protein Extraction PerformedSimultaneously with DNA and RNA Isolation...Anal Biochem(1999):192
2.Biotechniques. 1993 Sep ;15 (3):532-4, 536-7 7692896
A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.
Note: Protocol 1. Isolation of DNA and total RNA from single samples
Protocol 2. Isolation of RNA and Protein from same sample
Protocol 1. Isolation of DNA,RNA,and Protein simultaneously.
Protocol 2. Isolation of DNA,RNA,and Protein simultaneously (AllPrep DNA/RNA/Protein).
Protocol 3. Flow chart of isolation DNA,RNA,and Protein from same biomaterials.
Detailed procedure (click here)
Reference 1.Riol H, et al.Optimized Lymphocyte Protein Extraction PerformedSimultaneously with DNA and RNA Isolation...Anal Biochem(1999):192
2.Biotechniques. 1993 Sep ;15 (3):532-4, 536-7 7692896
A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.
Note: Protocol 1. Isolation of DNA and total RNA from single samples
Protocol 2. Isolation of RNA and Protein from same sample
DNA Extraction from Bacteria
Materials:Safety: Be careful working with Bacterial culture ! Wash hands! Procedure:
- E. Coli suspension (Luria Broth, 37oC overnight)
1% Sodium Dodecyl Sulfate (SDS)
Test tubes
Water Bath (60-65oC)
Stirring rod
95% Ethanol (chilled)
Dropper
- Add 5 ml. E. Coli suspension to a test tube.
- Add 1 ml. of SDS to the Tube with E. Coli, gently rotate and swirl to mix in SDS approx. 5 min.
- Stand tube in a 60-65o C water bath for 30 min.
- Allow to cool to room temp.
- Place a stirring rod into the test tube and add 2 ml. of cold 95% ethanol slowly with the dropper down the stirring rod into the tube.
- Hold tube at a 45 degree angle and slowly rotate your stirring rod clockwise (avoid touching sides of tube) approx. 5 min.
- Remove rod and immerse rod with bacterial DNA in 95% ethanol for approx. 2 min.
Begin working with DNA
Working with DNA
DNA is a big molecule, but it's still so small that there is no way to physically manipulate DNA.
It is all done chemically using enzymes like reverse transcriptase, polymerase chain reaction (PCR), sequence analysis, and restriction enzymes. Reverse transcriptase makes
DNA copies of RNA, PCR makes multiple copies of a particular gene and restriction enzymes
cut DNA at specific locations or nujcleotide sequences in a DNA molecule. These reactions
are all carried out in vitro without ever seeing a DNA molecule.
DNA storage: Genome DNA can be kept at 4 centigrade refrigerator, while we store plasmid DNA
and cDNA at -80 centigrade.
Concentration Measurement: DNA concentration (ul/ml)=OD260nmx50x(fold of dilution)
Purification: It should be pure enough for regular experiments, if the OD260nm/OD280nm is between
1.8-2.0
Video Show about DNA (click to reach the video source
DNA is a big molecule, but it's still so small that there is no way to physically manipulate DNA.
It is all done chemically using enzymes like reverse transcriptase, polymerase chain reaction (PCR), sequence analysis, and restriction enzymes. Reverse transcriptase makes
DNA copies of RNA, PCR makes multiple copies of a particular gene and restriction enzymes
cut DNA at specific locations or nujcleotide sequences in a DNA molecule. These reactions
are all carried out in vitro without ever seeing a DNA molecule.
DNA storage: Genome DNA can be kept at 4 centigrade refrigerator, while we store plasmid DNA
and cDNA at -80 centigrade.
Concentration Measurement: DNA concentration (ul/ml)=OD260nmx50x(fold of dilution)
Purification: It should be pure enough for regular experiments, if the OD260nm/OD280nm is between
1.8-2.0
Video Show about DNA (click to reach the video source