Molecular Biology Laboratory Techniques
DNA Cloning in a Plasmid Vector
DNA Cloning in a Plasmid Vector
Cloning DNA is an important technique in the molecular biology lab. Restriction enzymes and polymerase chain reaction (PCR) are used to obtain a specific segment of DNA. This segment of DNA is then “cut and pasted” into a plasmid vector. Plasmids are DNA that replicate independently of a cell’s chromosomal DNA – they are found in bacterial cells, though some animal cells may contain plasmids.
The short segment of DNA is joined with the plasmid vector’s DNA using an enzyme called DNA Ligase. Plasmid vectors contain three components: an origin of replication (ori), a gene for antibiotic resistance, and the cloning site (which contains the gene that will be cloned). Plasmids have approximately 3,000-12,000 base pairs, and are introduced into bacterial cells using a process called bacterial transformation.
The bacteria containing the cloned DNA then go through a process of cell screening: the cells are plated onto an antibiotic-laced agar. The bacteria containing the recombinant plasmids will be able to grow on these plates, since the plasmid contains a gene for antibiotic resistance, while the bacterial cells that did not take up the recombinant vector will die. The bacteria containing the vector will grow and divide, replicating the desired gene.
The bacterial cells are then broken down (a process called lysis) and the DNA is purified from the protein. Plasmids are fairly easy to isolate from chromosomal DNA because they are smaller – a process called fractionation is used to collect the plasmids.
Overview of Molecular Biology Techniques
- Vector Cloning: a segment of DNA is inserted into a plasmid. Bacterial cell growth and division creates many copies of the desired DNA sequence.
- PCR: Polymerase Chain Reaction allows for the replication and amplification of specific DNA sequences.
- Gel electrophoresis and SDS PAGE separate DNA or proteins into size-specific bands to allow for identification.
- Blotting (Western, Southern, etc.) allows for the detection and identification of DNA, proteins, or RNA.
- Microarrays allow for the detection of multiple areas of the genome in one test.
- HPLC and Chromatography purify proteins.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction is a method used in molecular biology laboratories to amplify short segments of DNA. PCR is the method used for performing "genetic fingerprinting" - paternity tests and DNA evidence collected from crime scenes are examples of the practical application of PCR.
DNA primers (which have the complementary sequence) are used, along with DNA Polymerase. If the desired sequence is ACTGACTG, for example, the primer would be TGACTGAC. The DNA polymerase used for PCR is usually Taq polymerase - a DNA polymerase with stability at high temperatures (its optimum performance is at 75-80°C).
Small volumes of a buffer containing the DNA are added to tubes in a thermal cycler. The solution is heated to approximately 95°C, where it is held for a few minutes. The DNA is then denatured at a high temperature (94-98°C): this causes the strands to separate, creating single stranded DNA. Denaturing DNA only takes a few seconds: this step usually requires about 30 seconds.
The DNA is then "annealed," which allows the primers to connect with the single strands of DNA. The temperature of the solution is lowered to approximately 55°C. The polymerase binds to the primer-template hybrid and starts to form the DNA.
The temperature is then increased to approximately 75°C (depending on the polymerase used), and the polymerase goes to work, creating DNA strands that complement the template. This continues, with each extension resulting in double the number of DNA strands. This leads to an exponential increase in the amount of DNA. At the end of the amplification process, the solution is held at 72°C for about 10 minutes to ensure that all single stranded DNA is elongated.
At the end of the PCR reaction, the solution is cooled to 4-8°C and stored. Gel electrophoresis is generally run to make sure the correct DNA sequence was amplified.
RT-PCR allows the amplification and quantification of DNA to occur at the same time. Real-time polymerase chain reaction detects the amount of DNA in real-time, as the chain reaction is occurring. This method is also used to detect mRNA: reverse-transcriptase is combined with the RT-PCR reaction and the amount of mRNA may be determined. Reporter probes are generally double stranded DNA coupled with a fluorescent marker. The fluorescence is measured and quantified during the reaction. RT-PCR is more accurate than Southern or Northern Blotting. This method is frequently used for diagnosing genetic abnormalities or cancer. RT-PCR is also used in research labs to detect changes in a gene's expression - for example, measuring the effect of a drug on a specific genetic sequence.
Nucleic acids (DNA and RNA) can be separated by size using a technique called gel electrophoresis. The nucleic acids are placed into wells in an agarose gel. An electric current is applied and the DNA/RNA migrates to the positive pole. The molecules are separated by length, as the shorter molecules of the nucleic acids move more quickly through the pores of the gel. A control sample with known nucleotide lengths (called a DNA Ladder) is used to infer the size of the bands. After gel used for electrophoresis is made with ethidium bromide, which makes the DNA bands visible under ultraviolet light. Since the ethidium bromide is not visible in natural light, a loading buffer containing bromophenol blue is usually used when injecting the sample into the well. This buffer will move at the same speed as the DNA, giving a visual indication of progress as the nucleic acid moves through the gel. A known sample is used to infer length of bands.
SDS PAGE 2D electrophoresis: similar to the gel electrophoresis method used for nucleic acids, proteins may be separated via electrophoresis. Since protein molecules are generally too small to get sieved by the larger gel pores, protein molecules are separated by charge. Another technique called SDS-PAGE is used to separate proteins by length. Polyacrylamide gel (PAGE) has uniform pore size and can separate proteins from 5-2,000 kDa (kilo daltons). Proteins are typically dyed with Coomassie Brilliant Blue stain.
Western Blot Video
Molecular Hybridization Techniques
Southern Blot is a way to view specific DNA sequences separated by gel electrophoresis. To verify that a specific gene has been obtained via PCR or vector cloning, the separated DNA is placed onto a filter, hybridized with a probe sequence, and visualized.
Named for biologist Edwin Southern, this test involves the placement of gel-electrophoresis separated single stranded DNA (ssDNA) onto a filter. A DNA hybridization probe is denatured to a single strand, then added to the filter. The probe attaches to the ssDNA target sequence. The probe contains a radioactive or fluorescent marker - the filter can be viewed by taking X-ray films (for radioactive markers) or under UV light (fluorescent markers).
Since the first test to study gene expression was called the Southern Blot after Edwin Southern, all other tests in the same general category are named in a corresponding fashion. The Northern Blot detects RNA in a sample rather than DNA. RNA is separated via gel electrophoresis, then placed on a Nylon membrane with a capillary blotting system. Heat is used to link the RNA to the membrane, then a labeled probe is hybridized to the RNA immobilized on the membrane. The RNA is then visualized using X-ray.
Using a similar technique to the aforementioned methods, Western Blot is used to detect proteins rather than DNA or RNA sequences. Gel electrophoresis is used to separate the proteins, which are then transferred to nitrocellulose. The nitrocellulose membrane is then "blocked" with a solution containing bovine serum albumin (BSA) and a small amount of Tween 20 (a detergent). Blocking prevents other, unwanted proteins from binding to the membrane.
Probes containing antibodies to the protein are added - the antibodies bind to the target protein. A secondary antibody is then used: the secondary antibody is linked to a color-reacting enzyme like horseradish peroxidase (HRP) and is targeted against the first antibody. When the secondary antibody binds to the primary antibody, a visible reaction takes place. The depth of the color determines how much protein is bound: a spectrophotometer is used to ojectively quantify the intensity of the colorimetric reaction.
An extension of the Western Blot, this technique detects post translational modifications (PTM's). When mRNA translates proteins, they typically undergo PTM before they are functional. The protein molecules may be glycosylated,sulfated, or modified in a myriad number of ways. The Eastern Blot detects the post-translational modifications that occur to proteins once they are translated from the mRNA.
Proteins are separated via SDS-PAGE and blotted onto a nitrocellulose mebrane. Probes specific to the type of modification are then added: if a researcher wants to view phosphorylation modifications to the protein, for example, she will use a probe specific to phosphorylation.
Similar to Eastern Blotting, Lectin Blotting probes for carbohydrate modifications to proteins and fats.
DNA microarrays are able to detect many regions of a genome with one test. Probe DNA is attached to a solid surface, targeting specific genetic sequences. The solid surface is either a silicon chip, glass, or microscopic beads. A sample is added to the plate and sequences that match the probes attach and bind to the probes. The chip is then washed to remove unbound DNA. The DNA is labeled with chemiluminescent targets, and the plate is read. The depth of the color development corresponds to the quantity of bound DNA.
Protein binding arrays are used to identify protein interactions, protein activation, and protein quantification in a patient sample. These arrays use glass or silicon as the solid surface, and typically use a monoclonal antibody to capture the target protein. Some protein microarrays use double stranded DNA specifically targeted to the protein as the capture mechanism. The amount of protein is quantified by the use of chemiluminescence, and the colorimetric reaction is measured on a microarray scanner.
These arrays are really miniaturized DNA microarrays - the test consists of tens of thousands of synthetic nucleotides on a solid surface. This high density test is frequently used for genetic screening: the Affymetrix oligonucleotide array, for example, is able to array more than 300,000 nucleotides in less than one square inch. Oligonucleotide arrays are used for genotyping, determining gene expression, mapping genomic libraries, and disease research.
Purification of Proteins and Peptides
There are several methods for purifying proteins in the microbiology lab. Once purified, proteins are concentrated by freeze-drying (lyophilization) or by the use of ultrafiltration, which concentrates the proteins on a membrane. Methods for purifying proteins include:
HPLC: High Performance Liquid Chromatography is a common technique used in molecular biology and biochemistry laboratories. High pressure drives the protein molecules up the hydrophobic column material. The proteins are then eluted from the column using a solvent, and the fractions of the desired protein are collected. The purified proteins may then be freeze dried for long term storage. HPLC is not used for proteins that lack the ability to re-fold - the high pressure generally causes the proteins to denature.
Resins coated with ligands specific to the desired protein are placed in a column. The protein binds to the ligand (which is often a lectin) and unbound proteins are washed away. A solution containing a high sugar content is then added to release the desired, bound protein: the sugar competes with the protein for the lectin binding sites. In some cases, the lectin must be denatured to release the bound protein. The purified protein is collected and stored.
Antibodies specific to the desired protein are coated onto column material. A sample solution is introduced to the column, and the targeted protein binds to the antibodies. Unbound proteins are washed away, and the desired protein is then eluted by changing the column salinity or pH.
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