BIOL 4125: Final Exam

List the ingredients required for a PCR reaction. Which ingredients can vary?

*1. dsDNA template with the "target sequence"* 2. Supply of four deoxyribonucleoside triphosphate (dNTP) *3. Heat stable DNA polymerase* *4. Two different ssDNA primers* 5. Buffer solution/Mg2+

What is the difference between PCR and DNA replication in E. coli in the following categories? Separate DNA: Starting Point: Primer: Product:

*E. coli*: Separate DNA: helicase and DnaA/DnaC Starting Point: oriC Primer: primase Product: leading/lagging strand *PCR*: Separate DNA: heat Starting Point: where you set primers Primer: synthetic primer Product: only leading strand

Compare and contrast Taq and Pfu polymerases.

*Taq:* Taq polymerase is a fast polymerase with good yield, producing maximum product. However, it has poor proofreading capabilities due to lack of DNA repair enzymes. As a result, it has a high misincorporation rate, and mistakes result in mutation. *Pfu*: Pfu polymerase is an example of a high-fidelity enzyme, so it has a much better proofreading capability than Taq DNA polymerase. As a result, it is good for amplifying DNA that will be cloned so the sequence synthesized is the same sequence as the original gene, although in lower yields.

What are the three steps of PCR? Describe each.

1. *Denaturation*: The reaction is heated to about 95 °C to denature the DNA into single-stranded DNA. 2. *Anneal*: The reaction temperature is reduced to about 45-68 °C to allow the primers to hybridize to their complementary sequences in the target DNA. 3. *Extension*: The reaction temperature is raised to 72 °C to allow Taq polymerase to synthesize DNA. *PCR is repeated for many cycles (about 25 to 30 cycles).*

What are the three characteristics that affect the migration rate of a molecule? Explain.

1. *Molecular Weight*: Small molecules migrate through pores and channels in the gel faster than large ones. 2. *Molecular Shape*: Tightly condensed globular molecules migrate more quickly than linear molecules. 3. *Molecular Shape*: Molecules with a greater negative charge will move toward the positive pole more quickly.

How does gel electrophoresis work?

1. A support matrix (the gel) is made, through which the molecules will move. 2. DNA is loaded into the sample wells at one end of the gel. The sample wells serve as the origin of migration. 3. The gel is filled with a buffer, such as TBE or TAE. 4. Once the samples are loaded, an electrical current is applied to the gel. 5. The samples migrate through tiny pores and passages in the gel matrix from the negatively-charged of the gel to the positively-charged end of the gel. 6. The gel is stained with ethidium bromide and exposed to UV radiation to view the DNA on the gel. SYBR green can be used as well. (2)

Describe transformation using electroporation.

1. All ions are removed from the cells by suspension in a nonconducting liquid such as de-ionized water. 2. DNA is added. 3. A very brief electrical shock at a high voltage of 1000-2000 V is applied, making the membrane porous and allowing DNA to enter the cell. 4. The cells are transferred to a new medium to recover from the shock and to undergo DNA replication. 5. The antibiotics genes, such as bla resistance genes, will be expressed in the plasmid. 6. The cells are plated on an antibiotic-containing plate, such as ampicillin, to select for the transformants.

How do you study a specific protein in bacteria?

1. Begin with your protein of interest. 2. Locate the gene that encodes for that protein of interest in the genome. 3. PCR to replicate DNA. 4. Gel electrophoresis is used to determine if the right gene was amplified by verifying protein size. 5. Clone the gene, or the PCR product, into a plasmid using restriction enzymes. 6. Digest the plasmid and perform gel electrophoresis again to confirm that cloning was successful. 7. Sequencing 8. Transformation, where the plasmid is inserted into a bacterial cell, occurs so that the bacterial cell itself will transcribe and translate into the protein product. 9. Protein expression is used to produce a large yield of the desired protein. 10. Protein purification

What are the three artificial transformation methods?

1. Cations and Heat Shock 2. Electroporation 3. Protoplast

Describe transformation using a protoplast.

1. DNA, Mg2+, and polyethylene glycol (PEG) are added to the cells. PEG modifies the membrane lipids and coats the DNA so that the DNA passes through the membrane. However, there is a problem: the peptidoglycan layer of the cell wall impedes the process. 2. Some of the cell wall is removed using lysozyme and penicillin treatments. Lysozymes break the β 1,4 glycosidic linkage between two adjacent sugars on one layer of peptidoglycan. Penicillin on the other hand inhibits transpeptidase. In other words, it prevents cross linking between two layers of peptidoglycan. The result is a spheroplast, where the cell wall is partially removed. 4. The cells are transferred to a new medium to recover from the shock and to undergo DNA replication. 5. The antibiotics genes, such as bla resistance genes, will be expressed in the plasmid. 6. The cells are plated on an antibiotic-containing plate, such as ampicillin, to select for the transformants.

What are four applications of PCR?

1. Forensics 2. Paternity Testing 3. Diagnosis of Diseases 4. Research

Describe protein expression.

1. Grow the transformed cells that contain the plasmid to the exponential phase so that the cell can produce the protein of interest at a high concentration. 2. Add isopropylthiogalactoside (IPTG), an allolactose analog. IPTG inactivates the repressor genes of the plasmid, allowing transcription of the genes of interest to produce a large amount of proteins. 3. Lyse the cells to extract the proteins using a lysis buffer, detergents such as SDS or Triton X, and lysozymes. 4. A small amount of the extracted protein is run on the SDS-PAGE to ensure the correct protein has been produced.

How do you lyse the cells during protein expression? What is the function of each component?

1. Lysis Buffer 2. Detergents such as SDS and Triton X: punch holes in the cell membrane 3. Lysozyme: rupture the cell wall

What are four things to avoid when designing DNA primers?

1. Primers should not exceed the range of 18-22 base pairs. 2. Two primers should not be complementary at the 3' end. 3. Primers should not have runs of three or more Gs or Cs at the 3' end of the primer. 4. Primers cannot be internally self-complementary.

What are three limitations of PCR?

1. Some knowledge of the target DNA sequences is required in order to determine primer sequences. 2. Amplification products longer than 10 kb are difficult to produce. 3. Minor contamination from other sources can cause problems.

What are the two buffers typically used in gel electrophoresis? What do they consist of?

1. TBE: Tris base, boric acid, and EDTA 2. TAE: Tris base, acetic acid, and EDTA

What are the two heat stable polymerases used in PCR?

1. Taq DNA polymerase from Thermus aquaticus 2. Pfu DNA polymerase from Pyrococcus furiosus

Describe transformation using cations and heat shock.

1. The addition of the cation such as Mg2+ or Ca2+ will neutralize the negative charge. 2. The DNA plasmid can now bind to the cell surface. 3. The cells with the DNA are heat-shocked at around 42 °C for about one minute. As a result, the cell membrane fluidity increases dramatically, creating pores in the membrane temporarily so that the DNA can enter the cell. 4. The cells are transferred to a new medium to recover from the shock and to undergo DNA replication. 5. The antibiotics genes, such as bla resistance genes, will be expressed in the plasmid. 6. The cells are plated on an antibiotic-containing plate, such as ampicillin, to select for the transformants.

In molecular biology, plasmids used for DNA cloning usually have been engineered to contain:

1. an origin of replication 2. a number of convenient restriction sites 3. a marker gene, such as antibiotics resistance genes, to select for its presence in the host cell

Plasmids may encode for:

1. drug and antibiotic resistance genes in pathogens 2. virulence factors in pathogens 3. bacteriocin 4. metabolic pathways that break down unique materials

Why is step three of PCR maintained at 72 °C?

72 °C is the optimal temperature for Taq polymerase.

What is a blunt end? Is it useful for cloning?

A blunt end is a straight cut through both strands of DNA at the line of symmetry. Blunt ends are not useful for cloning.

What is a spheroplast?

A cell in which the cell wall is partially removed.

In gel electrophoresis, what is a marker?

A marker is a set of standards that are used to determine the approximate size of a molecule run on the gel. A marker is needed when running a gel.

What is a vector?

A piece of DNA that can be used to artificially transfer foreign genetic material into another cell.

What is a plasmid?

A plasmid a small, circular extrachomosomal DNA molecule that replicates independently from the chromosomes within bacterial cells.

Why study a particular protein of interest in bacteria?

A protein's function, shape, and other characteristics can give insight and information into the organism itself.

What are the most commonly used materials for gel electrophoresis? What is the difference between them?

Agarose and polyacrylamide gels are the most commonly used for gel electrophoresis . Agarose is used for DNA and RNA while polyacrylamide is used for protein.

Who developed the first DNA sequencing protocols?

Allan Maxam and Walter Gilbert

What is bacteriocin?

Bacteriocin are proteinaceous toxins.

What is BLAST? What is it used for?

Basic local alignment search tool, or BLAST, is a program that allows the comparison of nucleic acid or protein sequences. It determines the percent similarity and difference between two sequences and identifies mutations.

During protein expression, why are cells that contain the plasmid cultured to the exponential phase?

Cells that contain the plasmid are cultured to the exponential phase so that the cells can produce the protein of interest at a high level and concentration.

Why is comassie brilliant blue dye used for SDS-PAGE?

Comassie blue is used for SDS-PAGE because this particular dye has a high affinity for proteins.

What is done with DNA restriction fragments?

DNA restriction fragments from the DNA to be cloned are added to the linearized vector in the presence of DNA ligase. As a result, a recombinant DNA is produced, which is then introduced into a bacterial host cell through the process of transformation.

What is the purpose of dideoxynucleotide DNA sequencing?

Dideoxynucleotide DNA sequencing is used to identify all of the nucleotides within a DNA sequence.

Describe Sanger sequencing.

Dideoxynucleotide DNA sequencing uses DNA polymerase to replicate new DNA from a single strand. Four aliquots are used. The four standard deoxynucleotide bases (dNTPs) are present in each aliquot. However, each aliquot has a small amount of one dideoxynucleotide (ddNTP), which lacks a 3' OH group. So, whenever a ddNTP is incorporated into the product DNA molecule, replication ceases. Each reaction tube produces a series of partial DNA molecules, each of which ends with that nucleotide. All four reactions must be run side by side on a gel in order to determine the complete sequence. The shortest bands are the DNA closest to the primer. These bands travel the fastest on the gel. The gel is read from the bottom up (5' end), all four lanes up.

What is EDTA?

EDTA is a metal ion chelator, which means it removes metals from nuclease, protecting DNA.

True or False: Transformation of E. coli is natural.

False

True or False: Genes in a plasmid are usually essential.

False: Genes in a plasmid are usually not essential, but they are usually beneficial.

True or False: PCR can still work even when there is a small amount of DNA present.

False: PCR only works when there is a large amount of DNA present.

True or False: PCR reactions are carried out in large volumes.

False: PCR reactions are carried out in small volumes (less than 100 μL).

True or False: Plasmids usually make up more than 5% of the overall genetic information in bacterial cells.

False: less

True or False: Both the plasmid DNA and PCR product to be cloned are cut with different restriction enzymes.

False: same

Why can't primers be internally self-complementary?

If primers are internally self-complementary, they will form hairpin loops.

Why can't primers be complementary at the 3' end?

If two primers are complementary at the 3' end, the primer dimer formation occurs where the primer is consumed, leading to low yields.

In general, what kind of DNA polymerase does PCR require? What has to be sacrificed to reach this goal?

In general, PCR needs a fast DNA polymerase to achieve good yields. However, proofreading activity, or a 3' to 5' exonuclease, would only slow down the synthesis rate. As a result, proofreading must be sacrificed for speed.

How do restriction enzymes provide a defense mechanism against foreign organisms, such as viruses?

Inside a bacterial cell, restriction enzymes selectively cut up foreign DNA. Host DNA is methylated by methylase to protect it from the restriction enzymes activity. However, since some viruses can produce methylase, this system is not 100% efficient.

What is IPTG? What is its function in protein expression?

Isopropylthiogalactoside (IPTG) is an allolactose analog that inactivates the repressor proteins of the genes on the plasmid, allowing transcription of the genes of interest during protein expression. This allows the cell to produce a large amount of proteins at a high level.

PCR was developed by ______________________________.

Kary Mullis

Why should primers not exceed the range of 18-22 base pairs?

Less than 18 base pairs is not specific enough of a primer while more than 22 base pairs will take too long to anneal to the DNA.

What is a lysozyme?

Lysozymes break the β 1,4 glycosidic linkage between two adjacent sugars on one layer of peptidoglycan.

What is the function of Mg2+ in PCR?

Mg2+ activates DNA polymerase.

What is the purpose of denaturing proteins during SDS-PAGE?

Most proteins have multiple subunits, so they may be too heavy for the gel. However, if the proteins are denatured by a detergent such as SDS, the motility of the protein on the gel increases.

Does PCR contain DNA gyrase or DNA ligase?

Nope!

Why must the DNA polymerase in PCR be heat stable?

PCR reaches temperatures upwards of 90 degrees. As a result, DNA polymerase must be heat stable and not denature.

What is the function of polyethylene glycol (PEG) in the formation of a protoplast?

PEG modifies the membrane lipids and coats the DNA so that DNA passes through the membrane.

What is the function of penicillin in the formation of a protoplast?

Penicillin inhibits transpeptidase. In other words, it prevents cross linking between two layers of peptidoglycan.

What is Pfu DNA polymerase? Where is it from?

Pfu DNA polymerase is from Pyrococcus furiosus, a hyperthermophilic archaea isolated from a deep sea thermal vent.

____________________________ is also known as artificial DNA replication.

Polymerase chain reaction (PCR)

What is the function of PCR primers?

Primers define the 5' and 3' boundaries of the replication products.

Describe the recognition sites for restriction enzymes.

Restriction enzymes recognize palindromic sequences. These recognition sites for restriction enzymes are usually 4-8 base pairs of double-stranded DNA.

What are restriction enzymes?

Restriction enzymes, or endonuclease, are "molecular scissors," found in bacteria and archaea species that fragment DNA at specific sites. They are thought to have evolved to provide as a defense mechanisms against foreign organisms, such as viruses.

How are restriction fragments produced?

Restriction fragments are produced when restriction enzyme cut the sugar-phosphate backbones of both strands of DNA.

Why should there not be runs of three or more G's or C's at the 3' end of a primer? Why are G's and C's such a concern? Why is the 3' end of the primer a concern?

Runs of three or more G's or C's at the 3' end of a primer reduces the specificity since homopolymer runs exists randomly in DNA templates. Gs and Cs are a concern because they form three hydrogen bonds versus two hydrogen bonds, creating a strong bond. The 3' end of the primer is a concern because Taq or Pfu DNA polymerase adds to the 3' end, so any error on the 3' end of the primer results in no PCR reaction.

What is SDS? What is its function during SDS-PAGE?

SDS is a detergent that denatures proteins and places a negative charge on the proteins. This allows all proteins to have a uniform negative charge so that they will be separated based on size on the gel.

Why must SDS place a negative charge on proteins during SDS-PAGE?

SDS places a negative charge on proteins so that charge is uniform and the proteins can be separated based on size.

What is SDS-PAGE? Describe each step.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, is a form of gel electrophoresis that is used during protein expression that ensures the correct protein has been expressed. SDS is a detergent that denatures the protein and places a negative charge on the proteins. The gel is stained with comassie brilliant blue dye. The gel is destained in water to the remove the excess stain, and the gel can then be viewed directly.

What are sticky ends? Are they useful for cloning?

Sticky ends are offset cuts that produce fragments with single-stranded overhanging ends. These overhanging ends can either be 5' or 3' overhangs. Sticky ends are ideal for inserting DNA and cloning.

What was the first heat stable polymerase used for PCR?

Taq DNA polymerase

What is Taq polymerase? Where is it from?

Taq DNA polymerase is from Thermus aquaticus, a bacterium isolated from Yellowstone hot springs. This is a heat stable polymerase that can be used in the PCR reaction.

How do you produce the "short product" in PCR?

Template DNA --> PCR --> Long Product --> PCR --> Short Product --> More Short Product

What method of DNA sequencing was preferred by 1977?

The Sanger dideoxynucleotide method was preferred by 1977.

What does the anneal temperature depend upon in PCR?

The anneal temperature in PCR depends on the sequence of the primer. For example, if there are more Gs and Cs in the primer than Ts and As, then the anneal temperature will be higher.

How are restriction enzymes named?

The first letter of a restriction enzyme is the genus from which the restriction enzyme is isolated. The next two letters of the restriction enzyme is the species name. The roman number reflects the order of discovery. For example, SmaI is isolated form Serratia marcescens and was discovered first.

What is the function of the cations in the usage of cation and heat shock for transformation?

The function of the cations is to neutralize the negative charge of the DNA and cell membrane so that DNA is able to pass through the membrane.

What is the function of the non-conducting liquid in electroporation?

The function of the non-conducting liquid in electroporation is to remove all ions from the cells.

What is the function of the heat shock step of transformation?

The heat shock increases cell membrane fluidity drastically, creating pores in the membrane temporarily that allow DNA to enter the cell.

What is a long product? How do you resolve the long product of DNA?

The long product is the gene of interest plus other DNA produced by PCR. The long product, however, is not desirable, as the gene of interest is all that is needed. The long product is resolved by a secondary round of PCR, where the gene of interest, also known as the "short product" is produced.

What is the problem that transformation must overcome?

The phosphate backbone of DNA and the cell wall each have a negative charge, so they repel each other. This charge difference must be overcome for transformation to occur.

Why is the protoplast method of transformation used?

The protoplast method of transformation is a last resort and used for cells that are resistant to other methods of transformation.

What serves as the origin of migration in gel electrophoresis?

The sample wells serve as the origin of migration.

What is meant by "exponential growth" in regards to PCR?

Through PCR, exponential growth of a target DNA sequence is possible. This mean that, with each round of PCR, the amount of DNA present doubles.

What is transformation?

Transformation involves transforming the plasmid DNA containing the PCR product into bacterial cells.

Which is the better polymerase: Taq or Pfu? Why?

Trick question: neither! A mixture of these two polymerases can be used to have the best of both worlds! A 9:1 Taq:Pfu DNA polymerase mixture is incredibly useful, as this mixture results in a good yield and good proofreading capability.

True or False: Bacteria can be naturally or artificially transformed.

True

True or False: Eukaryotes do not contain plasmids.

True

True or False: The higher the number of restriction enzymes, the better the plasmid.

True

Polymerase Chain Reaction (PCR)

an automated version of DNA replication that exponentially produces millions of copies of a short target DNA segment

Sanger sequencing was replaced by _______________________.

automated DNA sequencing

What do bla resistance genes encode for?

bla resistance genes encode for β lactamase, which cleaves β-lactam antibiotics.

Restriction enzymes produce restriction fragments with either ______________ or _______________ ends.

blunt; sticky

What is the safest form of transformation to use?

cation and heat shock

What is the most common form of transformation?

electroporation

With PCR, ____________________ growth of a target sequence is possible.

exponential

In the secondary round of PCR, the short product increases ____________________ while the long product increases __________________.

exponentially; geometrically

Gel Electrophoresis

procedure that allows the separation of molecules (proteins, DNA, or RNA) in an electric field based on differences in size, shape, and charge

What is the least common form of transformation?

protoplast

Why must the primers in PCR be ssDNA primers rather than RNA primers?

ssDNA primers are used over RNA primers because DNA is more stable.

Origin of Migration

the starting point of DNA in gel electrophoresis

PCR ingredients are mixed together and placed in a ______________________________, which controls temperature and time.

thermal cycler