DNA Isolation and Restriction Enzyme Analysis
The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced…
DNA extraction is the removal and purification of DNA from cells. First, cells are lysed - broken open - usually though a combination of physical disruption and treatment with chemicals, such a detergent which dissolves the cell and nuclear membranes. SDS or sodium dodecyl sulfate is a commonly use detergent that works by solubilizing the proteins and lipids that make up these membranes. The contents can then float freely into the surrounding solution.
Next, DNA must be separated from the other molecules present. Proteinase K added to the reaction will break down peptide bonds and digest contaminating proteins. Salt is then added to the sample, which stabilizes the negatively charged phosphate groups in the backbone of the DNA and, after the addition of ice cold alcohol, precipitates the DNA out of solution. The white precipitate is collected by spinning it in a centrifuge where it settles to the bottom of the tube. After washing and resuspending it in a buffer solution, the extracted DNA can finally be used in research or biotechnology applications.
Some biotechnology applications, like DNA fingerprinting, which can identify novel patterns in DNA specific to an individual or individuals, involve the use of restriction enzymes. Restriction enzymes are molecules which interact with DNA and recognize specific sequences. Once their specific site is identified, they cut the DNA. This will result in the strand being cut into one or more linear pieces. If the DNA extracted was a plasmid, a circular piece of DNA most often found in bacteria, any cuts will result in the DNA forming a linear fragment or fragments. Different restriction enzymes recognize different DNA sequences, so using a combination of these can result in distinct fragments being produced.
In DNA fingerprinting, we can then examine these fragments using a technique called DNA or gel electrophoresis. To prepare gels, powdered agarose is mixed with a buffer and heated until dissolved. A nucleotide stain is then added to the warm mixture and then this solution is poured into the casting mold. A comb is inserted to form the wells. When solidified, the gel is transferred to a gel box filled with buffer and the comb is removed. A reference mixture of dyed DNA fragments of known lengths, the DNA ladder, is added to one well and the dyed DNA samples of interest are loaded into the remaining wells. The box is connected to a power source and switching the power on induces the migration of the negatively charged phosphate groups in DNA nucleotides through the gel towards the anode, the positive end. Smaller pieces move more quickly than the larger fragments, which migrate with difficulty.
When the run is complete the gel is exposed to ultra violet light to visualize the nucleotide stain in the DNA samples. Their presence can be confirmed based on their relative location to the ladder bands. Because DNA with different sequences will have cut sites at different locations, this can produce novel band patterns, or fingerprints, which can be used to distinguish individuals or variant DNA profiles.
In this lab you will perform DNA extractions using a buffer with and without SDS to assess the importance of detergents in DNA isolation and then digest plasmid DNA with different restriction enzymes to examine the resulting DNA profiles.
DNA extraction is the removal and purification of DNA from cells. First, cells are lysed - broken open - usually though a combination of physical disruption and treatment with chemicals, such a detergent which dissolves the cell and nuclear membranes. SDS or sodium dodecyl sulfate is a commonly use detergent that works by solubilizing the proteins and lipids that make up these membranes. The contents can then float freely into the surrounding solution.
Next, DNA must be separated from the other molecules present. Proteinase K added to the reaction will break down peptide bonds and digest contaminating proteins. Salt is then added to the sample, which stabilizes the negatively charged phosphate groups in the backbone of the DNA and, after the addition of ice cold alcohol, precipitates the DNA out of solution. The white precipitate is collected by spinning it in a centrifuge where it settles to the bottom of the tube. After washing and resuspending it in a buffer solution, the extracted DNA can finally be used in research or biotechnology applications.
Some biotechnology applications, like DNA fingerprinting, which can identify novel patterns in DNA specific to an individual or individuals, involve the use of restriction enzymes. Restriction enzymes are molecules which interact with DNA and recognize specific sequences. Once their specific site is identified, they cut the DNA. This will result in the strand being cut into one or more linear pieces. If the DNA extracted was a plasmid, a circular piece of DNA most often found in bacteria, any cuts will result in the DNA forming a linear fragment or fragments. Different restriction enzymes recognize different DNA sequences, so using a combination of these can result in distinct fragments being produced.
In DNA fingerprinting, we can then examine these fragments using a technique called DNA or gel electrophoresis. To prepare gels, powdered agarose is mixed with a buffer and heated until dissolved. A nucleotide stain is then added to the warm mixture and then this solution is poured into the casting mold. A comb is inserted to form the wells. When solidified, the gel is transferred to a gel box filled with buffer and the comb is removed. A reference mixture of dyed DNA fragments of known lengths, the DNA ladder, is added to one well and the dyed DNA samples of interest are loaded into the remaining wells. The box is connected to a power source and switching the power on induces the migration of the negatively charged phosphate groups in DNA nucleotides through the gel towards the anode, the positive end. Smaller pieces move more quickly than the larger fragments, which migrate with difficulty.
When the run is complete the gel is exposed to ultra violet light to visualize the nucleotide stain in the DNA samples. Their presence can be confirmed based on their relative location to the ladder bands. Because DNA with different sequences will have cut sites at different locations, this can produce novel band patterns, or fingerprints, which can be used to distinguish individuals or variant DNA profiles.
In this lab you will perform DNA extractions using a buffer with and without SDS to assess the importance of detergents in DNA isolation and then digest plasmid DNA with different restriction enzymes to examine the resulting DNA profiles.
Q1: Why is SDS used during DNA extraction?
SDS, or sodium dodecyl sulfate, is a detergent that dissolves the lipids and proteins making up cell and nuclear membranes. By solubilizing these membrane components, SDS allows the cell contents, including DNA, to float freely into the surrounding solution, making DNA accessible for further purification steps.
Q2: What role does Proteinase K play in DNA isolation?
Proteinase K is an enzyme that breaks down peptide bonds in proteins, digesting contaminating proteins like histones that bind to DNA. This separation is essential because DNA is tightly condensed around histone proteins inside the nucleus, and removing these proteins allows pure DNA to be recovered.
Q3: How does salt help precipitate DNA from solution?
Salt, typically sodium chloride, stabilizes DNA by allowing Na+ ions to bind to and integrate into the negatively charged phosphate groups in the DNA backbone. This causes DNA strands to clump together, making the precipitate visible and collectible by centrifugation.
Q4: What determines where restriction enzymes cut DNA?
Restriction enzymes recognize and cut DNA at specific nucleotide sequences, typically six to twelve nucleotides long and usually palindromic, meaning they read the same in both the 3'-5' and 5'-3' directions. Different restriction enzymes recognize different sequences, allowing researchers to produce distinct DNA fragments for analysis.
Q5: Why do smaller DNA fragments move faster through gel electrophoresis?
During gel electrophoresis, negatively charged DNA migrates toward the anode through the gel matrix. Smaller DNA fragments navigate the gel's pores more easily than larger fragments, which experience greater resistance and migrate with difficulty, causing size-based separation of DNA pieces.
Q6: How can restriction enzyme digestion create unique DNA fingerprints?
Because different DNA sequences have cut sites at different locations, digesting DNA with restriction enzymes produces novel band patterns specific to each individual's genetic profile. These unique patterns, visualized through gel electrophoresis, can distinguish individuals or variant DNA profiles for forensic and genetic analysis.
Q7: What happens to plasmid DNA when restriction enzymes cut it?
Plasmids are circular DNA molecules commonly found in bacteria. When restriction enzymes cut a plasmid at their recognition sites, the circular DNA becomes linear, forming one or more linear fragments. These fragments can then be isolated and ligated into vectors for bacterial transformation using plasmids procedure applications.