Topic 6: Molecular Genetics Flashcards
Phenol-Chloroform Method (DIY Method)
- Works with all organisms and tissues
- The phenol-chloroform is important because it helps to separate the soluble DNA and the soluble cell debris in the later steps.
The phenol-chloroform method is a common technique used in molecular genetics to isolate DNA from cells or tissues. This method involves using a mixture of phenol and chloroform to extract DNA from the sample, followed by precipitation and purification of the DNA. This method involves the following steps:
- Homogenize (aka grind) or suspend cells, and harvest cells or tissue containing DNA.
- Burst the cells (e.g., via detergent) which disrupts the cells or tissue to release DNA.
- Digest cell components, through acidification (acetic acid, or salt) to the disrupted sample and mix well.
- Centrifuge the sample to separate the aqueous (top) layer from the organic (bottom) layer (i.e., we get solid debris at the bottom and an aqueous solution at the top - RECALL that DNA and RNA are soluble in water therefore, the DNA and RNA will be located in the aqueous component)
- Collect the aqueous layer, which contains the DNA, and transfer it to a new tube (this is called the supernatant)
- Add phenol-chloroform to the supernatant; the phenol-chloroform will dissolve soluble debris.
- Centrifuge the sample again.
- Take the aqueous/supernatant layer, which is comprised of the DNA and RNA, and transfer it to a new tube.
- Wash the DNA with ethanol to remove any remaining impurities, the ethanol will cause the DNA to precipitate out of the water.
- Centrifuge the sample again, which will cause a DNA pellet.
- Resuspend the DNA in a buffer or water for further use.
Homogenize (aka grind)
Homogenization is a process that is often used in molecular genetics to break down cell or tissue samples into smaller, more uniform pieces prior to DNA extraction. The process involves physically disrupting the sample using a homogenizer, blender, or other mechanical means, such as grinding or crushing.
Supernatant
In DNA extraction, the supernatant is typically the aqueous layer that is collected after centrifugation following the addition of phenol-chloroform to the sample. The aqueous layer contains the DNA, RNA, and other water-soluble components of the sample, while the organic layer contains lipids, proteins, and other hydrophobic molecules. After centrifugation, the aqueous layer is collected and further processed to isolate and purify the DNA.
Column Based DNA Extraction
- more expensive, but easier to use
- Designed for model organisms
This method involves passing the sample through a specialized column that contains a resin or membrane with affinity for DNA. The DNA binds to the column material while other components of the sample are washed away, allowing for the purification of the DNA.
The steps involved in column-based DNA extraction can vary depending on the specific kit or protocol being used, but generally involve the following:
- Homogenize or suspend
- Burst the cells (i.e., Lysis of cells or tissues to release DNA)
- Transfer the liquid containing the burst-up cells on top of the column (the column is a test tube that acts as a filter, a silica filter that is designed to bind DNA) - DNA binds to the column material.
- Wash away impurities and other components of the sample that did not bind to the column material. Via centrifuge of the sample with the column, adding a buffer to dissolve different components. The DNA will get stuck to the silica gel.
- Elution of the purified DNA from the column using water. Soak in water, and the DNA will dissolve off of the silica membrane and go into the water. In the end, you will get pure DNA and water.
Lysis of Cells
Lysis of cells refers to the process of breaking open or disrupting cell membranes to release the contents of cells. This is an important step in many molecular biology techniques, including DNA extraction, because it allows access to the DNA molecules within the cells.
There are many methods for cell lysis, which vary depending on the type of cells being lysed and the downstream application. Commonly used lysis methods include physical disruption (such as sonication or grinding with a mortar and pestle), enzymatic digestion (such as with proteinase K or lysozyme), and chemical disruption (such as with detergents or organic solvents).
Plasmid Extraction: Mini Prep
Steps 1-4: growing and collecting bacteria;
Step 5: Harvesting of bacterial cells- The bacterial cells are harvested by centrifugation, and the resulting bacterial pellet is resuspended in a buffer solution.
Step 6: Lysis of bacterial cells: The bacterial cells are lysed using a detergent-based lysis solution that breaks open the bacterial cell wall and releases the plasmid DNA. And add an alkali buffer (basic) solution; basic solutions cause the DNA to separate (from double-stranded to single-stranded)
Step 7: Add a buffer to neutralize the cells (bring the pH back down), and the DNA will reanneal (i.e., go back to being single-stranded). They anneal for only a short time (this leaves behind the genomic DNA and allows the plasmid DNA to reanneal.
Step 8: Wash steps: The spin column is washed with one or more buffers to remove contaminants, salts, and other impurities that could interfere with downstream applications.
Step 9: Elution of plasmid DNA: The plasmid DNA is eluted from the column using a low-salt buffer, water, or a specialized elution buffer, resulting in highly purified plasmid DNA.
In Plasmid Extraction: Mini Prep, the goal is to isolate and purify plasmid DNA from a bacterial culture. The procedure involves disrupting the bacterial cells to release their contents, including both the genomic DNA and plasmid DNA. Then, the plasmid DNA is selectively captured and purified using a specialized column that binds to the plasmid DNA but not the genomic DNA or other cellular components. The resulting purified plasmid DNA can be used for various downstream applications, such as cloning, gene expression studies, and gene editing.
It’s important to note that plasmid DNA is usually much smaller than genomic DNA, which can make it easier to isolate and purify. Additionally, plasmid DNA often carries specific genetic elements that can be manipulated for research purposes, making it a valuable tool in molecular biology.
What is the difference between genomic DNA and plasmid DNA?
Genomic DNA and plasmid DNA are two types of DNA found in cells, and they have distinct characteristics and functions.
Genomic DNA is the DNA that makes up the chromosomes of a cell. It contains the complete genetic information of an organism, including both coding and non-coding regions. Genomic DNA is present in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotic cells.
Plasmid DNA, on the other hand, is a small, circular, double-stranded DNA molecule that is separate from the genomic DNA. Plasmids are found in many bacteria and some eukaryotic cells, and they can replicate independently from the genomic DNA. Plasmids often carry genes that provide selective advantages to the host cell, such as antibiotic resistance or the ability to degrade certain compounds.
Polymerase Chain Reaction (PCR)
- PCR is a method of synthesizing DNA in vitro
- Basically, we put DNA polymerase in a test tube with everything it
needs to synthesize DNA - Polymerase Chain Reaction (PCR) is a laboratory technique used to amplify a specific DNA sequence.
- The result of the PCR process is that each cycle doubles the amount of DNA that is produced, leading to a rapid and exponential increase in the number of copies of the target DNA sequence.
in vitro
In vitro refers to a scientific process or experiment that takes place outside a living organism in a controlled laboratory setting. It literally means “in glass” in Latin, as experiments are often conducted in glass containers like test tubes, petri dishes, or flasks.
DNA polymerase
DNA polymerase is a type of enzyme that plays a crucial role in DNA replication and repair. It catalyzes the polymerization of deoxyribonucleotides, the building blocks of DNA, into a new DNA strand complementary to a preexisting DNA template strand.
During DNA replication, DNA polymerase moves along the template strand and adds nucleotides to the growing complementary strand according to base pairing rules (A-T and C-G). This process ensures that the newly synthesized DNA strand is identical to the original template strand, thereby maintaining the genetic information encoded in the DNA.
What is required in PCR:
- DNA Template: Any type of DNA that you are working with; can be from your extraction or any other source. You need to know about the DNA sequence that you are working with (necessary so you can design primers)
- Primers that complement the template (i.e., primers that are 17-25 nucleotides long complement the template) - work the same way that DNA synthesis (requires an open 3’ OH group). We design these primers.
- Forward Primers (upstream of the target)
- Reverse Primers (downstream of the target)
- dNTPs: Nucleotide triphosphates (DNA building blocks)
- MgCl2 = Mg is a cofactor of DNApol
- Buffer solution = waters + salts (creates an environment that is similar to the inside of a cell)
Why do we need a forward and reverse primer in PCR?
In PCR, a forward and reverse primer are used to initiate DNA amplification and define the region of DNA that will be copied. The forward primer binds to one end of the target DNA sequence, while the reverse primer binds to the other end of the target DNA sequence in the opposite orientation.
The reason for using both a forward and reverse primer in PCR is to ensure that only the desired DNA fragment is amplified. When the reaction mixture is heated during the denaturation step of each PCR cycle, the double-stranded DNA template is separated into two single strands. During the annealing step, the forward and reverse primers anneal to their complementary sequences on the template DNA strands, which then serve as the starting points for DNA synthesis by the DNA polymerase.
By using primers that are specific to the target DNA sequence, PCR can amplify only the desired fragment of DNA and avoid amplifying other regions of the genome. The specificity of PCR is important in many applications, including gene cloning, DNA sequencing, and medical diagnosis.
PCR Steps
Polymerase chain reaction (PCR) involves a series of temperature-dependent steps that amplify a specific segment of DNA. The basic steps of PCR are as follows:
- Denaturation: The reaction mixture is heated to a high temperature (usually around 95°C) to denature the double-stranded DNA into single strands. For approx. 30-60 secs, this will separate the two strands of DNA
- Annealing: The temperature is then lowered to allow the primers to anneal to their complementary sequences on the single-stranded DNA template. The annealing temperature is typically around 50-60°C, depending on the primers used. For approx. 30-60 secs, which is long enough for the primers to get annealed onto the DNA template
- Extension: The temperature is raised again to around 72°C to activate the DNA polymerase, which synthesizes new DNA strands from the primers. The DNA polymerase adds nucleotides to the primers in the 5’ to 3’ direction, extending the new DNA strand. The extension time depends on the length of the DNA fragment being amplified but typically ranges from 30 seconds to 2 minutes.
- Repeat: The denaturation, annealing, and extension steps are repeated for a set number of cycles (usually 25-35 times), with each cycle doubling the amount of DNA produced. The number of cycles is dependent on the starting amount of DNA and the desired amount of amplified product.
- exponentially increase the amount of target DNA in the sample
- 2^n copies of DNA are made, with n=number of cycles
What innovations were required for PCR?
- DNA polymerase from Thermus aquaticus (Taq) was discovered; this is a DNA polymerase that is stable at high temperatures. Found in bacteria that live in boiling hot springs in Yellowstone park. Before this, you had to add a polymerase at each step.
- Development of automated thermocyclers (prior, we had to have three different baths of water, and we had to manually move the tube); this cycles through the temperatures as needed.
Gel electrophoresis
Gel electrophoresis is a laboratory technique used to separate and analyze DNA, RNA, or proteins based on their size and charge. It involves placing a sample of the biomolecule mixture onto a gel matrix, applying an electric field across the gel, and allowing the biomolecules to migrate through the gel matrix. The gel matrix acts as a sieve, separating the biomolecules according to their size and charge.
During gel electrophoresis, the biomolecules are loaded into wells at one end of the gel and an electrical current is applied to the gel. The biomolecules migrate through the gel towards the opposite end, with smaller molecules moving faster than larger molecules. This separation allows researchers to visualize and analyze the different components of the biomolecule mixture.
- Helps to visualize DNA
- Gel electrophoresis can be used to separate DNA molecules on the basis of their size.
- Gel is made from agarose.
- DNA molecules that are exposed to an electric field migrate toward the positive pole due to the negatively charged phosphates along the DNA backbone.
- Since the charge-to-mass ratio for all DNA is the same, it is the shape of DNA molecules that determines the rate of migration.
- DNA is run on agarose or polyacrylamide gels
Ethidium bromide
It intercalates between the bases of nucleic acids and fluoresces red-orange (560nm) in UV light (260-360nm). Glows in UV light, and is used in PCR so we can see the DNA in the gel.
- Causes mutations (able to get into cells)
SYBR-green (SYBER-Safe)
Forms a complex with DNA that absorbs 497 nanometers of blue light (λmax = 497 nm) and emits green light (λmax = 520 nm). Supposedly safer than Ethidium bromide. Glows when we shine a particular wavelength on it.
Why do we compare our samples to “Ladders” in PCR?
In PCR, ladders are used as a reference to determine the size of the amplified PCR product. A DNA ladder is a mixture of DNA fragments of known lengths that are run alongside the PCR samples on an agarose gel during gel electrophoresis. The DNA ladder appears as a series of bands on the gel, with each band representing a DNA fragment of a specific size.
By comparing the size of the bands in the ladder to the size of the bands in the PCR samples, it is possible to determine the size of the amplified PCR product. This is important for verifying that the correct product has been amplified and that the reaction was successful.
In addition to size determination, ladders can also be used to determine the approximate amount of DNA in the PCR sample, by comparing the intensity of the PCR bands to the intensity of the bands in the ladder. This can help to ensure that the PCR reaction was carried out with the correct amount of starting material and that the reaction was not inhibited or contaminated.
Restriction Enzymes (RE) / Endonucleases
- Enzymes that cut dsDNA at specific sequences called restriction sites
- Naturally occurring enzymes in bacteria
- Bacteria use DNA endonucleases called restriction enzymes to destroy bacteriophage (i.e., viruses that infect bacteria) chromosomes
- Bacteriophages are viruses that infect and destroy bacteria
- We want to work with restriction ends that create sticky ends
Bacteria use DNA endonucleases called restriction enzymes (RE) to destroy bacteriophage chromosomes: How do they do this, and what sequences are they looking for?
Bacteria use DNA endonucleases called restriction enzymes to destroy bacteriophage (the virus that infects bacteria) chromosomes by cleaving their DNA.
- Restriction enzymes (RE) recognize and cut specific DNA sequences, known as restriction sites or recognition sites. The REs look for specific sequences.
- These restriction sites are usually palindromic, meaning that they read the same in both directions, and typically consist of 4 to 8 base pairs.
- Hind-III recognizes the sequence shown in the image. Whenever Hind-III encounters a piece of DNA it is going to become associated with that piece of DNA and is going to start to look for the sequence: AAGCTT
- Sequences are palindromic, meaning that it reads the same in forward and reverse
Palindromic
In the context of molecular biology, a palindromic sequence refers to a sequence of nucleotides that reads the same on both strands of double-stranded DNA when read in the opposite direction. This means that the sequence is symmetrical and can be read the same way from left to right or right to left on both strands of DNA.
Palindromic sequences are important in many biological processes, such as DNA replication, recombination, and restriction enzyme recognition sites. For example, some restriction enzymes recognize and cut palindromic sequences, which can be useful for molecular biology techniques such as DNA cloning and gene editing.
An example of a palindromic sequence is the DNA sequence 5’-GATATC-3’, which reads the same on both strands when read in the opposite direction: 5’-GATATC-3’ (top strand) and 3’-CTATAG-5’ (bottom strand).
Hind-III
A straggered cutter, it will cut between the A’s in the sequence AAGCTT, which creates “sticky ends.”
- Sticky ends are ideal, we want REs that make sticky ends.
Sticky Ends
when restriction enzymes cut DNA at a specific recognition sequence, they can create overhangs called “sticky ends.”
Sticky ends are overhanging single-stranded regions of DNA that are created when the restriction enzyme cuts the DNA at an offset position within its recognition sequence.
The overhangs can be complementary to the overhangs of another DNA fragment that has been cut with the same restriction enzyme. This complementary base pairing enables the fragments to anneal, or hybridize, together and can be used to create recombinant DNA molecules.
The creation of sticky ends is important in genetic engineering because it allows researchers to join two DNA fragments together. The sticky ends can be used to make a stable, specific, and directional connection between two DNA fragments.
Sticky ends can also be used to insert DNA fragments into plasmids or other vectors for transformation into bacteria or other organisms. The overhangs of the DNA fragment and the vector can be joined together using DNA ligase, which seals the nick between the two molecules, creating a recombinant DNA molecule.
Restriction enzymes do not cut their own host DNA because of:
- Methylation hides the RE site from the enzyme but is only added to the host cell chromosome
- Therefore any foreign DNA with this sequence will be cut by the RE
- In the image, EcoR1 is looking for the sequence shown, but the host is hiding them by attaching CH3 (Methylation)
How do we create Recombinant DNA
- Cut the DNA fragments with restriction enzymes: Digest the DNA fragments with the chosen restriction enzymes to create sticky ends.
- Mix the DNA fragments: Combine the purified DNA fragments that have complementary sticky ends and incubate them together.
- Anneal the sticky ends: The complementary sticky ends of the DNA fragments will base-pair with each other, creating a stable hybrid molecule.
- Ligating the DNA fragments: Use a DNA ligase enzyme to covalently link the two fragments together.
- Transforming the recombinant DNA: Introduce the recombinant DNA into a suitable host cell, such as bacteria or yeast, where it can be replicated and expressed.
How do we conduct Hybridization
This is how we find DNA among a sample that complements a known piece of DNA, we do this through a process called “probing.”
- Obtain the probe: The probe is a single-stranded nucleic acid molecule that is complementary to the target sequence. It can be either DNA or RNA, depending on the target sequence being studied. Probes are labeled with a reporter molecule (such as a radioactive or fluorescent tag) for detection.
Add the probe to the sample: The probe is added to the sample containing the denatured target DNA or RNA, and allowed to hybridize or base-pair with the complementary target sequence. - Wash the sample: The sample is washed to remove any unbound probe molecules that did not hybridize with the target sequence.
- Detect the hybridized probe: The presence of the bound probe is detected using a suitable detection method, depending on the label used on the probe (such as autoradiography, fluorescence, or chemiluminescence).
Sanger Sequencing (Sanger dideoxynucleotide or chain-terminating DNA sequencing)
Uses: DNA extraction, PCR, AND gel electrophoresis (i.e., we combine all three); best for sequencing one or a couple of genes
- Four PCR reactions (contains all usual components of PCR), BUT THEN we add special dNTPs
- Each reaction contains the normal
components PLUS a small amount of one
of four dideoxynucleotides (ddATP, ddCTP,
ddGTP, or ddTTP).
- If the dideoxynucleotide is incorporated into
the synthesized strand, DNA synthesis
stops.
Example PCR Gel: what can you infer?
A PCR (Polymerase Chain Reaction) gel is a type of gel electrophoresis that is used to visualize and analyze the amplified DNA fragments that are generated by PCR.
When we amplify a different gene in PCR and look at the results, we can look to see if there are different lengths of that gene within the species.
In this example, we see that there are two different lengths: there are two different alleles of this gene:
- 412 bp (longer)
- 336 bp
What is special about the Dideocyribonucleoside triphosphate (ddNTP) USED for Sanger DNA Sequencing? Why is it important?
Rather than an “OH” we have an “H” - they will not be able to produce an open 3’ OH group when they get incorporated into the DNA strand that is being produced, when they get incorporated DNA synthesis stops on that particular strand.
The key difference between ddNTPs and dNTPs is the absence of the 3’-OH group in ddNTPs. This is important because the 3’-OH group is required for the formation of a phosphodiester bond between the 3’-end of the incoming nucleotide and the 5’-end of the previous nucleotide in the growing DNA chain during DNA synthesis. Without the 3’-OH group, ddNTPs cannot participate in chain elongation, resulting in premature termination of the DNA chain.
Sanger Sequencing (Sanger dideoxynucleotide or chain-terminating DNA sequencing) STEPS
What are the nucleotides colour-coded as? In Sanger Sequencing
T = Red
A = Green
C = Blue
G = Black
Missense Mutation
a missense mutation is a genetic mutation that changes a single nucleotide in the DNA sequence, resulting in the incorporation of a different amino acid in the protein sequence, which can have varying effects on the structure and function of the protein.
a missense mutation does not change the length of the DNA strand. This is because a missense mutation involves a single nucleotide substitution in the DNA sequence, which means that the overall length of the DNA strand remains the same.
Frameshift Mutation
A frameshift mutation is a type of genetic mutation that results in the addition or deletion of one or more nucleotides in the DNA sequence, which can cause a shift in the reading frame during protein synthesis. This shift can alter the amino acid sequence of the resulting protein, often resulting in a truncated, non-functional, or completely different protein product.
Frameshift mutations change the length of the DNA strand, as they add or delete one or more nucleotides from the sequence.
Southern Blot STEPS
- Start with an unknown sample of DNA: This is the first step in any Southern blot. The sample can be genomic DNA or any other DNA source, as long as it contains the sequence of interest.
- In southern blots, we always start with an unknown sample of DNA (e.g., genomic DNA (gDNA) extraction) - Digest the DNA with a restriction enzyme: Restriction enzymes are used to cut the DNA into smaller fragments, which will allow us to identify the sequence of interest. It’s important to choose the correct restriction enzyme for the DNA being used.
- Then digest the gDNA with a restriction enzyme (i.e., cutting up the gDNA sequence) - Run on a gel: Gel electrophoresis separates the DNA fragments by size, with smaller fragments moving faster through the gel than larger fragments.
- Run on the gel (i.e., gel electrophoresis) - Transfer DNA fragments: After gel electrophoresis, the DNA fragments need to be transferred from the gel to a membrane such as nitrocellulose or nylon. This can be done through capillary action or by using an electric current.
- Transfer DNA fragments - Denature the DNA: Denaturation of the DNA involves separating the double-stranded DNA into single strands. This is typically done using an alkaline solution.
- Denature to get DNA fragments from double-stranded to single-stranded DNA fragments (denature is done via alkaline solution) - Hybridization with a labelled probe: A labelled probe is a short strand of ssRNA or ssDNA that is complementary to the sequence of interest. It will hybridize or bind to the single-stranded DNA on the membrane, allowing us to identify the sequence of interest.
- Where the probe sticks will help us find us determine what the unknown DNA sequence is
- We now know that our gene of interest is in the black band of DNA (referring to the image)
How to READ Souther Blots
Cloning STEPS
- Obtain the source DNA: The first step is to obtain the DNA sequence of interest from a biological sample. This can be genomic DNA, plasmid DNA, or any other DNA source that contains the sequence of interest.
- Select restriction enzymes: Choose a restriction enzyme that will cut the source DNA sequence at a specific site to produce the desired fragment for cloning. It’s important to ensure that the chosen restriction enzyme does not cut within the sequence of interest.
- Digest the DNA with restriction enzymes: Incubate the DNA sample with the chosen restriction enzyme to cut the DNA sequence at the specific site. This will produce the desired fragment for cloning.
- Prepare the cloning vector: The cloning vector is a DNA molecule used to carry the DNA fragment of interest. It is often a plasmid that has been modified to contain a selectable marker, such as antibiotic resistance, and unique restriction sites for inserting the DNA fragment.
- Digest the cloning vector with the same restriction enzymes: Cut the cloning vector with the same restriction enzymes used to digest the source DNA. This will create sticky ends that are complementary to the ends of the DNA fragment and allow for the two pieces to be ligated together.
- Ligate the DNA fragment into the cloning vector: Mix the digested DNA fragment and cloning vector together with DNA ligase, which catalyzes the formation of covalent bonds between the two pieces of DNA. This creates a recombinant DNA molecule containing the source DNA sequence within the cloning vector.
- the last circular component in the image, is the recombinant DNA
How to determine/selection of transformed bacteria:
Steps 1-4: getting our foreign DNA into a plasmid and making recombinant DNA
- need to create markers that tell us that our bacteria has taken up plasmids; we are going to have three options:
- Bacteria with no plasmids (will NOT grow)
- Bacteria with an empty plasmid (will grow, but do not have foreign DNA and will be BLUE)
- Bacteria with a recombinant plasmid (will grow, and will be WHITE)
