Molecular Biology Techniques

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What is needed for a basic PCR set-up?

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- DNA template that contains the DNA region (target) to be amplified.
- Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
- TAQ polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
- Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
- Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
- Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7]
- Monovalent cation potassium ions.

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What are the steps in a basic PCR?

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1. Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.[9]
2. Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.
3. Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA formation .
4. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,[10][11] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
5. Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
6. Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

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What is RT-PCR?

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Reverse Transcription PCR (RT-PCR): for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends).

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Western Blot

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In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis.

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Gel Electrophoresis

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The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel.

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Polyclonal Antibodies

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Polyclonal antibodies bind to multiple epitopes of a given antigen and are produced by (1) immunizing a mammal – often a goat or rabbit – with an antigen, (2) bleeding the animal periodically, and (3) extracting the antibodies directly from the serum.1 The process is relatively inexpensive, and large quantities of an antibody can be isolated from a single extraction.

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Monoclonal Antibodies

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monoclonal antibodies are derived from a single (‘mono-’) clone of antibody-producing B cells. After immunizing a mammal, the animal’s spleen (location of B cell production) is extracted, and the B cells are fused to immortal myeloma cells; the fused B cells and myeloma cells – or hybridomas – are screened via ELISA, and the selected hybridoma line is injected into a second mammal. The myeloma component of the hybridoma induces localized tumor growth in the animal, and an antibody-rich fluid called ascites can be extracted from the tumor; the antibodies are then isolated themselves through column chromatography.2 A powerful feature of monoclonal antibodies is that the immortal nature of the hybridoma allows for its continual reculturing, thus providing a constant source of monoclonal antibodies.

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ELISA

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1. A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.
2. A solution of non-reacting protein, such as bovine serum albumin or casein, is added to block any plastic surface in the well that remains uncoated by the antigen.
3. Next the primary antibody is added, which binds specifically to the test antigen that is coating the well. This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen.
4. Afterwards, a secondary antibody is added, which will bind the primary antibody. This secondary antibody often has an enzyme attached to it, which has a negligible effect on the binding properties of the antibody.
5. A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme. The color change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. This can be helpful in a clinical setting, and in R&D.
6. The higher the concentration of the primary antibody that was present in the serum, the stronger the color change. Often a spectrometer is used to give quantitative values for color strength.

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Electroporation

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is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance inside the cell, such as loading it with a molecular probe, a drug that can change cell's function, or a piece of coding DNA.

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Transformation

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involves the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign DNA.

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Origin of Replication

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is a unique DNA sequence at which DNA replication is initiated. DNA replication may proceed from this point bidirectionally or unidirectionally.

The specific structure of varies somewhat from species to species, but all share some common characteristics. This site binds a member of the pre-replication complex—a protein complex that binds, unwinds, and begins to copy DNA.

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Promoter

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DNA sequences in concert with the proteins they help recruit.  They designate where to start copying a gene. Are known as the promoter and transcription initiation site.

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What do you need in a good expression vector?

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1. strong promoter
2. inducible promoter (drug or heat inducible)
3. ribosome binding site near an ATG codon

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Selection vs. Screening

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selection is when only the gene you want will grow (e.g. when you clone by complementation)

screening is when many things grow and you have to find your gene by a probe

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PCR, briefly

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1. denature at 95 degrees
2. anneal primers at 50 degrees
3. elongate strandfs at 72 degrees.
4. repeat 20-35 times.

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Reporter Gene

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a gene attached to a promoter or translation start site, and used to measure the activity of the resulting transcription or translation. The reporter gene serves as an easily assayed surrogate for the gene it replaces.

e.g. lacZ, cat

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Restriction Enzyme Digest

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Enzyme bind DNA at specific sequence Cleave it and cuts DNA in a particular fashion creating "STICKY ENDS" which can rehybridize with eachother or other DNA cut with same enzyme

Enzyme analysis can be used to ID DNA
Can be used to recombine DNA

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What is one thing a PCR absolutely has to have?

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A primer! Primers are designed to hybridize to DNA flanking target sequence. 2 primers are used so that both strands of DNA are replicated.

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Why Does a Western Blot use 2 Antibodies?

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Primary antibody-unique to protein
Secondary antibody- recognizes constant region of primary antibody and binds to it.

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DNA sequencing

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1. A primer sequence is determined for an unknown fragment of single-stranded DNA. 

2. This unknown DNA is combined with DNA polymerase, primer, dNTPs and ddNTPs which have been marked with fluorescent dye. If a dNTP is incorporated into the growing strand, replication continues. If a ddNTP is used, however, replication is terminated. 

3. Replication is allowed to continue until many different fragments of varying lengths are produced. These fragments are separated by gel electrophoresis. The terminating ddNTP is identified by its color, and the sequence can be "read" from the bottom of the gel to the top. 

4. The sequence of the newly synthesized DNA (which is deduced from the gel) is the complement of the unknown strand.

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Gene Knockout

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-allows testing of gene function, and produces mouse models of human genetic disease.

1. targeted gene on a plasmid inactivated by insertion of a marker gene 

2. vector inserted into a mouse stem cell 

3. recombination puts the marker/inactivated target in chromosome 

4. stem cell transplanted into an early mouse embryo 

5. extensive selection and further matings can give knock out mouse

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Gel Electrophoresis

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Gel electrophoresis separates DNA molecules based on their size and electrical charge. A porous gel is made from agarose (a polysaccharide from seaweed), which is melted in a buffer solution and poured into a mold. Wells at one end hold the samples, and an electric current passes through the gel. The DNA fragments go to the positive end of the gel because of their negative phosphate groups, and they separate by size (smallest go furthest).

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Gene Cloning

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Gene cloning is when you amplify a specific piece of DNA by placing the piece in a bacterial cell and letting it replicate the DNA.

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Cloning Vector

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A cloning vector is a stable, replicating DNA molecule that a foreign DNA piece can attach to for its introduction into the cell.

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What does a good cloning vector need to have?

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A good cloning vector needs three characteristics: (1) an origin of replication that makes sure the vector is replicated in the cell, (2) selectable markers, which let any cells with the vector be found or selected, and (3) one or more unique restriction sites where a DNA fragment can be inserted.

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Plasmids

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Plasmids are circular DNA molecules in bacteria, and they are common vectors for cloning DNA in bacteria.

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Inserting a gene into a plasmid vector

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The easiest way to put a gene into a plasmid vector is to cut the foreign DNA (containing the gene) and the plasmid with the same restriction enzyme, making cohesive ends so that the plasmid and the foreign DNA get mixed together.

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Selectable Markers

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Selectable markers are on plasmids, and they let the cells with recombinant plasmids be detected.

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DNA Palindromes

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recognition sequences can be read from either strand when read in the 5' to 3' direction

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How to create recombinate DNA and transform into ecoli

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1. isolation of DNA to be copied, treatment w/ enzyme to make fragments

2.  fragments ligated to plasmids cut with same enzyme - recombinant vector

3.  vector transferred to E. coli when replicates 

4.  bacteria plated on nutrient medium, form colonies, screened for presence of recombinant plasmids

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Genomic Library

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contains at least one copy of all the sequences in  a genome, constructed using host cell cloning since PCR fragments are small

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10 Essential Amino Acids

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PVT TIM HALL

P= Phenylalanine
V= Valine
T=Threonine
T=Tryptophan
I=Isoleucine
H=Histidine
A=Arginine
L=Leucine
L=Lysine

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Amino Acids with Hydrophobic Side Chains

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Alanine
Isoleucine
Leucine
Valine
Phenylalanine
Tryptophan
Tyrosine