Lesson 7: DNA Technologies

Sanger sequencing

A method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication

(C) 1,048,5762^20 = 1,048,576. You will have 1,048,576 double-stranded DNA fragments after the 20th cycle of PCR. To know how many fragments of DNA after any cycle simply calculate 2 raised to the cycle's number (2^n).

After the first cycle of PCR, you have 2 double-stranded DNA fragments. After the second cycle, you have 4. And after the third cycle, you have 8. How many will you have after the 20th cycle? (A) 248,642 (B) 486,922 (C) 1,048,576 (D) 1,854,392

(D) in vivo, in vitro DNA Cloning is done in vivo (in a living thing such as a bacteria) while PCR is done in vitro (in a test tube).

DNA Cloning is done _______________ while PCR is done ________________. (A) in vitro, in vitro (B) in vitro, in vivo (C) in vivo, in vivo (D) in vivo, in vitro

(D) II and III Only DNA Cloning requires the use of DNA Ligase and Restriction Enzymes.

DNA Cloning requires the use of which of the following enzymes? I. DNA Polymerase II. DNA Ligase III. Restriction Enzymes (A) I Only (B) II Only (C) I and II Only (D) II and III Only

Because the Sticky Ends share exposed, matching base pairs, these should ligate more easily.

Draw out both Sticky and Blunt ends. Which do you expect to ligate more easily?

Restriction Enzymes

Enzyme that cuts DNA at a specific sequence of nucleotides

(C) intercalating agent Ethidium bromide is a common example of an intercalating agent.

Ethidium bromide is a common example of a(n): (A) reducing agent (B) biological agent (C) intercalating agent (D) oxidizing agent

Cellular contents are injected into each well. If mRNA from the cell is located in that well, the mRNA from the cell will hybridize to the mRNA in the well, and it will not get washed out. This shows the researcher that that specific mRNA's gene is expressed in that cell.

Explain how microarray experiments depend on hybridization.

Size

Gel electrophoresis sorts DNA molecules on the basis of their?

Gene knockout

Genetic manipulation in which one or more of an organism's genes are prevented from being expressed

Negative end, due to the fact that it has a negative charge, so it would be pulled to the positive end

In gel electrophoresis, what end does the DNA start?

Smaller

In gel electrophoresis, will the smaller or larger particles migrate further?

You could extract the mRNA that produces the virus's capsid (outer layer). Then you could produce cDNA using reverse transcriptase, and insert that cDNA into a cloning host such as e Coli. Then extract the protein and inject it into humans. This would allow the human's immune system to build immunity against that virus without the risk of getting the disease.

Originally, vaccines entailed injecting people with a virus in the hopes that they would develop immunity against that virus. The problem with this is that the vaccine might actually give you the disease. How was DNA technology used to overcome this obstacle?

B)8 segments, with the 2 original strands on different segments

PCR (polymerase chain reaction) is an excellent method of generating copies of target DNA. If a single piece of double stranded DNA (dsDNA) is put into a PCR machine, how many dsDNA segments will there be after 3 rounds? A)8 segments, with the 2 original strands paired B)8 segments, with the 2 original strands on different segments C)16 segments, with 2 original strands on different segments D)16 segments, with the 2 original strands paired

False. Each band represents tons of DNA strands of the same length. If there was only one DNA strand, it would be impossible to see it.

True or False? Each band that you see during DNA gel electrophoresis represents a single DNA strand.

False. Transgenic Mice expressing the phenotype of the Recombinant Vector in all of their cells must have had parents with the Recombinant Vector in their Germ Cells, or the Recombinant Vector was added to embryonic stem cells.

True or false? Transgenic Mice expressing the phenotype of the Recombinant Vector in all of their cells must have had parents with the Recombinant Vector in all of their cells too.

1. Target DNA molecule 2. A pair of DNA primers 3. Heat-resistant DNA polymerase 4. All 4 dNTPS (A,C,G,T)

What are the 4 things needed for PCR?

D)Gel Electrophoresis Polymerase Chain Reaction uses specially constructed primers to amplify a specific DNA sequence. Thus, you can tell if the target sequence was in the sample based on if the reaction produces more nucleic acid. A Southern blot is one of the most efficient method for detecting the presence of a specific DNA sequence in a sample. It works via hybridization of the target sequence to a probe. If the probe latches on, the target is there. Gel Electrophoresis separates fragments based on size, and so is not nearly as specific as the other techniques mentioned above.

Which procedure is least effective for detecting a specific DNA sequence in a sample? A)Microarray B)Southern Blot C)Polymerase Chain Reaction D)Gel Electrophoresis

Many bacteria in the petri dish will not take in the plasmid. We want these bacteria to die so that we know we only have bacteria on our plate that contain the gene of interest. To kill off all bacteria without a plasmid, we add an antibiotic. Only bacteria that contain the plasmid will survive since the plasmid contains an antibiotic resistance gene that makes them immune to that specific antibiotic.

Why do the plasmids used in DNA Cloning contain an antibiotic resistance gene?

Adding an intercalating agent (such as ethidium bromide) allows us to physically see the DNA sample. The agent works by placing itself between the nucleotide base pairs of the DNA and becomes fluorescent when you apply UV light to it. This allows us to visualize the DNA and thus determine how far it traveled in the gel.

Why is adding an intercalating agent (such as ethidium bromide) an important step in the process of running gel electrophoresis?

The acidic solution lilkely affected the charge on the DNA due to the protonation of phosphate groups. This affected the rate at which the DNA migrated towards the positive electrode.

Working on a research project, you run a gel electrophoresis on some DNA samples. Subsequently, you find out from your principal investigator that your DNA samples have been damaged during the gel electrophoresis. You find out that the buffer solution in which you bathed the DNA was acidic. Why did this damage your DNA and ruin your gel electrophoresis?

Since each protein has the same pKa, each protein will travel the same distance on the gel. In Isoelectric Focusing, a protein stops moving when the pH of the gel is equal to its isoelectric point, which would be the same for each of these proteins.

You conduct Isoelectric Focusing on three proteins with the same pKa but differing molecular weights of 250 Daltons, 100 Daltons, and 25 Daltons. Which will travel the furthest in the gel?

The police may use PCR to create many copies of two different regions of your genome known to contain a variable number of repeats (known as Short Tandem Repeats (STRs)). They would then run that DNA using gel electrophoresis to measure the size of the DNA (the more repeats, the less far it will migrate). If the tested fragments from hair DNA migrate to the same locations as the tested fragments from your DNA, they'd pin you as the criminal!

You just robbed a bank, and a piece of your hair was found at the scene of the crime. GASP! You are taken into custody as a suspect, and your DNA is tested against the DNA found in the hair originally discovered. How might DNA technology be used by the police to prove that you are guilty?

Southern blot

A DNA sample is electrophoresed on a gel and then transferred to a filter. The filter is then soaked in a denaturant and subsequently exposed to a labeled DNA probe that recognizes and anneals to its complementary strand. The resulting ds labeled piece of DNA is visualized when the filter is exposed to film. What is this?

Taq polymerase

A DNA synthesis enzyme that can withstand the high temperatures of PCR

Binary Fission

A form of asexual reproduction in single-celled organisms by which one cell divides into two cells of the same size

B)A petri dish completely overrun with untransformed and transformed bacteria Antibiotics kill bacteria - they are not part of the transfection process. Adding antibiotics is a vital step that allows scientists to selectively grow only the bacteria that carry the antibiotic-resistant gene that was transfected into them. Bacteria divide very rapidly. By the time the scientist came in to check on his petri dish, it would have been covered in a lawn of transformed and untransformed bacteria.

A scientist is trying to express gene X in bacteria. After cloning the cDNA from gene X into a plasmid with the appropriate cDNA, and transfecting the plasmid into bacteria the scientist notices that he forgot to add antibiotics. What will greet him the next day? A)A mostly normal petri dish - antibiotics are just used as a safe-guard against other bacteria B)A petri dish completely overrun with untransformed and transformed bacteria C)A petri dish with separate colonies of transformed and untransformed bacteria D)A petri dish with only untransformed bacteria, the antibiotics are required for transfection

D)Polymerase Chain Reaction

A student adds Taq Polymerase, nucleotides, and primer to a test tube. What procedure are they most likely going to do next? A)Southern Blot B)Gel Electrophoresis C)Microarray Hybridization Assay D)Polymerase Chain Reaction

D)The student forgot to add reverse transcriptase

A student was attempting to make cDNA that would code for protein Y. They began with an isolated and amplified sample of the target post-transcriptional mRNA, and all the tools required to complete the task. However, at the end of the experiment the student was left with a molecule containing ribonucleotides. What most likely went wrong? A)The student forgot to add DNA polymerase B)The student added RNA polymerase instead of DNA polymerase C)The cloning vector already transcribed the DNA D)The student forgot to add reverse transcriptase

Northern blotting

A technique that enables specific nucleotide sequences to be detected in samples of mRNA. It involves gel electrophoresis of RNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe.

Reverse Genetics seeks to understand the phenotype via the alteration and/or composition of the genotype (as in knock out experiments). Forward Genetics, on the other hand, seeks to understand the genotype simply by observing the natural phenotype (as in observing bean plants and their shapes and sizes.

Compare Reverse Genetics with Forward Genetics.

Genomic DNA libraries contain larger fragments of double-stranded DNA and include both exons (coding regions) and introns (noncoding regions).cDNA libraries only contain an organism's expressed genetic information in the form of double-stranded cDNA.

DNA libraries consist of either genomic DNA or cDNA (complementary DNA). What is the difference between genomic libraries and cDNA libraries?

Recombinant DNA

DNA that has been formed artificially by combining constituents from different organisms.

(1) Cut - Restriction Enzymes are used to cut the DNA into smaller pieces.(2) Gel Electrophoresis - DNA fragments are placed in wells and will travel from one end of the gel to the other, getting separated based on their size (note that the Khan Academy video incorrectly states that the fragments are separated based on size and charge, but they will only be separated based size since the mass-to-charge ratio is equal between fragments).(3) Filter Paper - Filter paper is place on the gel, and the fragments will transfer over to the filter paper. (4) Probe - A radio-labelled single-stranded DNA fragment is added to the filter paper. It will hybridize to the gene of interest.(5) X-ray - In order to visualize the radio label, the filter paper is exposed to an x-ray, which will only show a band for the gene of interest.

Describe each of the 5 steps of a Southern Blot: (1) Cut(2) Gel Electrophoresis(3) Filter Paper(4) Probe(5) X-ray

(1) Cut - Restriction Enzymes are used to cut the gene of interest out of a greater body of DNA.(2) Paste - That gene is then inserted into a plasmid. (3) Transform - The plasmid is placed in a solution with bacteria. Heat is applied, which causes the bacteria to take in the plasmid. This uptake process is termed "transformation." (note that Khan Academy incorrectly uses the term "transformation" to describe the Paste step.(4) Amplify - The bacteria are grown on a plate, increasing the amount of the gene of interest.

Describe the four steps of DNA Cloning:(1) Cut(2) Paste(3) Transform(4) Amplify

(1) PCR - PCR is used to amplify the gene of interest.(2) PCR with ddNTPs - The sample is separated into four different containers, one containing regular nucleotides plus radio-labelled ddGTP, another container with ddCTP, another with ddTTP, and another with ddATP. PCR is then continued in each of the four containers.(3) Gel Electrophoresis - The sample is place in a well and the DNA strands travel across the gel, becoming separated by size.(4) Analysis - The DNA strands are analyzed in order from shortest to longest fragment.

Describe the steps of DNA Sequencing: (1) PCR(2) PCR with ddNTPs (3) Gel Electrophoresis(4) Analysis

(1) Denaturation - The double stranded DNA that you are interested in studying is denatured as the temperature is increased to 96° C. (2) Annealing - As the temperature is decreased to 55° C, the primer anneals to the end of the region that you want to copy on each strand of DNA. (3) Extension - DNA Polymerase (which is most active at a temperature of 72° C) begins extending the DNA from the primer.

Describe what happens during each step of PCR and why the temperature is set to what it is during each step: (1) Denaturation (96° C) (2) Annealing (55° C) (3) Extension (72° C)

(D) All of the Above

Gel Electrophoresis is used to figure out the size and charge of macromolecules such as: (A) DNA (B) RNA (C) Protein (D) All of the Above

Dideoxynucleotides (ddNTPs) lack 3' OH group typical of regular deoxynucleotides (dNTPs). Because they lack this OH group, ddNTPs cause DNA polymerization to stop since the phosphate head of the downstream nucleotide can't bind to an H group.

How do dideoxynucleotides (ddNTPs) differ from typical deoxynucleotides (dNTPs) in terms of structure and function?

B) The ddNTP has no 3' hydroxyl group, so no condensation reaction can occur to add another nucleotide

How does the addition of a ddNTP prevent further elongation of the DNA strand? A) The ddNTP is fluorescently labeled, preventing further DNA polymerase action B) The ddNTP has no 3' hydroxyl group, so no condensation reaction can occur to add another nucleotide C) The ddNTP has no 2' hydroxyl group, so no condensation reaction can occur to add another nucleotide D) The ddNTP causes a hairpin loop to form, causing the DNA polymerase to dissociate with the DNA strand

We can isolate mRNA from a cell and use the enzyme reverse transcriptase to generate single-stranded cDNA using the mRNA as a template. This cDNA represents the original template strand's exons.

How is cDNA generated?

Instead of using restriction enzymes to isolate DNA in step 1, you would use Reverse Transcriptase to generate cDNA templates of the cell's mRNA. Then you would make that cDNA double-stranded via DNA Polymerase. This double-stranded DNA can then be pasted into the plasmid and the process continues as normal from step 2 onward.

How would these steps differ if you wanted to clone cDNA?

DNA Polymerase sythesizes the complimentary strand, making the cDNA double-stranded and able to be inserted into a cloning vector for a cDNA library.

If cDNA is single-stranded, how can it be inserted to a cloning vector that makes up a cDNA library?

You can conclude the following:- Gene A's well has no color. This gene is not expressed in either the normal or cancerous cell.- Gene B's well has a red color. This gene is expressed primarily in the cancerous cell. Perhaps this gene's protein is contributing to the cancer.- Gene C's well has a yellow color. This gene is expressed equally well in both the normal and cancerous cell.- Gene D's well has a green color. This gene is primarily expressed in the normal cell. Perhaps the lack of this gene's protein is contributing to the cancer.

In a certain microarray experiment, the mRNA in the regular cell is labelled with a green flouresent tag while the mRNA in the cancer cell is labelled with a red flouresent tag. When red and green are mixed, they produce a yellow color. What can you conclude regarding the following genes based on the results of the microarray:- Gene A's well has no color. - Gene B's well has a red color.- Gene C's well has a yellow color.- Gene D's well has a green color.

The shortest fragments are located at the bottom of the gel, and DNA is synthesized in the 5' to 3' direction. Thus, the sequence of the DNA is Red, Red, Yellow, Red: 5'-TTGT-3'

In a particular DNA sequencing experiment, ddGTP was labelled yellow, ddCTP was labelled blue, ddTTP was labelled red, and ddATP was labelled green. What is the sequence of the DNA strand from 5' to 3' if the gel displayed the following from top to bottom:RedYellowRedRed

B)Bacteria from viruses

In nature, restriction enzymes are meant to protect A)Humans from bacteria B)Bacteria from viruses C)Bacteria from other bacteria D)Viruses from bacteria

Eukaryotes: Introns & Exons Prokaryotes: Exons

In relation to introns/exons, what do prokaryotes and eukaryotes contain?

The anions always move toward the anode while the cations always move toward the cathode in an electrical field.

In what direction do the anions and cations move towards in an electrical field?

You would place samples of DNA fragment on the negative electrode end because DNA is negatively charged due to its phosphate backbone, you would want the DNA to travel across the gel towards the positive electrode.

On which side of the gel electrophoresis (negative electrode or positive electrode) would you place samples of DNA fragment?

Amplification of the DNA/cDNA is accomplished through inserting the cloning vector into a bacteria that can replicate that genetic material over and over again. From there, we can sequence the gene of interest.

Once DNA or cDNA is inserted into a cloning vector, how is that genetic material "amplified"?

(C) III > I > II The polymerase chain reaction (PCR) happens subsequently in the following order: (1) Denaturation(2) Annealing(3) Extension

Place the steps of a polymerase chain reaction (PCR) in the correct order: I. Annealing II. Extension III. Denaturation (A) III > II > I (B) II > III > I (C) III > I > II (D) I > II > III

Gel electrophoresis

Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel

(C) II > IV > I > III The steps of DNA Cloning are as follows: (1) Cut(2) Paste(3) Uptake(4) Amplify

Put the following steps of DNA Cloning in order from first to last: I. Uptake II. Cut III. Amplify IV. Paste (A) I > III > II > IV (B) III > I > II > IV (C) II > IV > I > III (D) II > IV > III > I

(A) II, I, IV, III, V The steps of a Southern Blot are as follows:(1) Cut(2) Gel Electrophoresis(3) Filter Paper(4) Probe(5) X-ray

Put the following steps of a Southern Blot in the correct order from first to last: I. Gel Electrophoresis II. Cut III. Probe IV. Filter Paper V. X-ray (A) II, I, IV, III, V (B) II, I, IV, V, III (C) I, III, V, IV, II (D) II, IV, I, III, V

(B) IV > II > I > III The steps of DNA Sequencing are as follows: (1) PCR(2) PCR with ddNTPs (3) Gel Electrophoresis(4) Analysis

Put the steps of DNA Sequencing in the correct order: I. Gel Electrophoresis II. PCR with ddNTPs III. Analysis IV. PCR (A) II > IV > I > III (B) IV > II > I > III (C) I > III > I > IV (D) III > I > IV > I

The special traits in how restriction enzymes work are by recognizing palindromic sequences (meaning that the 5' to 3' sequence of one strand is identical to the 5' to 3' sequence of the other strand in antiparallel orientation) and by producing offset cuts yielding sticky ends on the fragments.

Restriction enzymes (restriction endonucleases) work by recognizing what types of sequences?

Restriction map

Shows the lengths of DNA fragments between restriction sites in a strand of DNA

Microarray

Silicon or glass sheet with thousands of DNA probes that can be used to identify which genes in a tissue are expressed

Sticky ends

Single stranded ends of DNA left after cutting with enzymes

Plasmids

Small rings of DNA found naturally in some bacterial cells in addition to the main bacterial chromosome. Can contain genes for antibiotic resistance, or other "contingency" functions.

Polymerase chain reaction (PCR)

Technique that allows molecular biologists to make many copies of a particular gene/DNA

Western blotting

Technique that uses antibodies to detect the presence of specific proteins separated by electrophoresis

Exponentially

The PCR cycles increase?

Cathode

The negatively charged electrode end is considered the?

Anode

The positively charged electrode end is considered the?

DNA sequencing

The process of determining the precise order of nucleotides within a DNA molecule

DNA hybridization

The process used to compare structural characteristics of organisms to determine how closely related there are

I. Southern - (B) DNA strands presentII. Northern - (D) mRNA levelsIII. Eastern - (A) Post-Translational Protein modificationsIV. Western - (C) Protein levelsI like to think of these as a bizarro-compass rose. Instead of "North South East West", I think of these as "South North West East".

There are four directional types of Blots in general. Match the type of blot with the information it provides. I. Southern II. Northern III. Eastern IV. Western (A) Post-Translational Protein modifications (B) DNA strands present (C) Protein levels (D) mRNA levels

Insulin and Human Growth Hormone

These were produced through recombinant DNA technology and used in medicine to help patients

The benefits of agarose gel (polyacrylamide gel) are that it allows a complete protein to be recovered after an analysis since SDS is a denaturant and can damage DNA or denature proteins, and secondly the agarose gel (polyacrylamide gel) determines the globular size of proteins.

What are the relative benefits of agarose (polyacrylamide gel) compared to sodium dodecyl sulfate (SDS)-polyacrylamide gel?

The benefits of sodium dodecyl sulfate (SDS)-polyacrylamide gel is that it can be used to eliminate charge as a factor and focus specifically on molecule's size.

What are the relative benefits of sodium dodecyl sulfate (SDS)-polyacrylamide gel compared to agarose (polyacrylamide gel)?

Denaturation (96°C), primer annealing (55°C), primer extension (72°C)

What are the steps of PCR?

PCR, ddNTP addition, & gel electrophoresis

What are the steps to DNA sequencing?

Termination of strand elongation

What does ddNTP terminate?

Number of DNA molecules double

What happens after one cycle of PCR?

DNA ladder is a set of standardized DNA molecules of known size that are used to determine the size of an unknown DNA sample molecule run on a gel during electrophoresis.

What is a DNA ladder and why do we need one when running a DNA gel electrophoresis?

DNA library is a collection of the total genomic DNA from a single organism that have been cloned into vectors. With access to such a library, researchers can identify and isolate the DNA fragments that interest them for further study.

What is a DNA library and what is its purpose?

Agarose gel (polyacrylamide gel electrophoresis) is used for larger DNA fragments and the mobility of DNA fragments in this gel depends on both size and charge. Sodium dodecyl sulfate (SDS)-polyacrylamide gel is used for smaller macromolecules (either small DNA or small proteins) and the mobility through this gel strictly depends on size alone since SDS disrupts all noncovalent interactions. Also, SDS will cause the protein to unfold.

What is the difference between the two most common electrophoresis types: agarose (polyacrylamide gel electrophoresis) vs. sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis?

Plasmid can't be too large in size

What is the one limitation to recombinant DNA using plasmids?

(A) Agarose gel Agarose gel is the preferred gel for DNA electrophoresis.

What is the preferred gel for DNA electrophoresis called? (A) Agarose gel (B) Polysaccharide gel (C) Sucrose gel (D) Polyacrylamide gel

The purpose of PCR is to generate tons and tons of copies of a single fragment of DNA that you are interested in. This is essential for researchers since it would be very hard to work with just a single copy of DNA since they are so tiny and fragile. Having many copies allows you to better test and visualize the DNA you are interested in.

What is the purpose of Polymerase Chain Reaction (PCR)?

Conducting a Southern Blot allows the researcher to determine if a specific gene is present within a larger body of DNA.

What is the purpose of a Southern Blot?

The purpose of Gel Electrophoresis is to figure out the size and charge of macromolecules such as DNA, RNA and proteins.

What is the purpose of gel electrophoresis?

Single stranded DNA

What kind of DNA is cDNA?

(B) I and II Only Isoelectric focusing and ion-exchange chromatography both separate proteins based on charge, which can indirectly separate proteins based on their pI.

What separation method(s) can be used to isolate a protein on the basis of the isoelectric point? I. Isoelectric focusing II. Ion-exchange chromatography III. SDS-PAGE (A) I Only (B) I and II Only (C) II and III Only (D) I, II, and III

The smaller, digested fragments would be able to re-bond with their original molecules, decreasing the efficiency of the ligation.

What would happen if a researcher skipped the Gel Electrophoresis in between the restriction digest and ligating?

cDNA libraries have genes that can be expressed in a cloning host to produce functioning proteins. This is due to the fact that the DNA is "pre-spliced" (introns are already removed). Genomic libraries cannot be used to produce functioning proteins since the host (typically bacteria) that proteins are produced in do not contain spliceosomes, and thus cannot splice out introns.

Which DNA library (genomic library or cDNA library) has genes that can be expressed in a cloning host to produce functioning proteins?

Genomic libraries have promoter and enhancer sequences present, but not necessarily in the same clone.cDNA libraries do not have these these regions present since they would not be found within the exons of the gene.

Which DNA library (genomic library or cDNA library) has promoter and enhancer sequences present?

The DNA with 3500 base pairs will travel further in a gel electrophoresis because gel acts like a sieve, thus allowing small particles to travel through more easily while retaining large particles; consequently the shorter the DNA then the further it can travel through the gel.

Which DNA would travel the farthest in a gel electrophoresis? A DNA with 6000 base pairs or DNA with 3500 base pairs?

(D) I, II and III Each of the following statements are true:I. cDNA Libraries will lack all introns typically removed by alternative splicing.II. cDNA Libraries can vary based on the type of cell the RNA was taken from.III. cDNA Libraries isolated from one cell/cell type can be called Expression Libraries.

Which of the following statements about cDNA libraries are true? I. cDNA Libraries will lack all introns typically removed by alternative splicing. II. cDNA Libraries can vary based on the type of cell the RNA was taken from. III. cDNA Libraries isolated from one cell/cell type can be called Expression Libraries. (A) I only (B) I and II only (C) II and III only (D) I, II and III

(D) Both the insert and the cloning vectors are digested with restriction enzymes, providing Sticky Ends. To prevent those cleaved sequences from recombining with their smaller fragments, the restriction digest products undergo Gel Electrophoresis, and the largest band is extracted from the gel. Then, the insert and vector are ligated together.

Which of the following statements best describes how most sequences are inserted into cloning vectors? (A) Both the insert and the cloning vectors are digested with restriction enzymes, providing Blunt Ends, and then the two are ligated together. (B) Both the insert and the cloning vectors are digested with restriction enzymes, providing Blunt Ends. To prevent those cleaved sequences from recombining with their smaller fragments, the restriction digest products undergo Gel Electrophoresis, and the largest band is extracted from the gel. Then, the insert and vector are ligated together.(C) Both the insert and the cloning vectors are digested with restriction enzymes, providing Sticky Ends, and then the two are ligated together. (D) Both the insert and the cloning vectors are digested with restriction enzymes, providing Sticky Ends. To prevent those cleaved sequences from recombining with their smaller fragments, the restriction digest products undergo Gel Electrophoresis, and the largest band is extracted from the gel. Then, the insert and vector are ligated together.

(C) The vector is linearized as 1 fragment. Think of this like making one cut in the band of a wedding ring. It would no longer be an intact circle shape, but all of the metal would still be connected!

Which of the following would happen if you restriction digested a vector at exactly one Restriction Site? (A) 2 fragments form (B) The Vector is linearized and 2 fragments form (C) The vector is linearized as 1 fragment. (D) None of the above.

(B) The Vector is linearized and 2 fragments form

Which of the following would happen if you restriction digested a vector at exactly two Restriction Sites? (A) 2 fragments form (B) The Vector is linearized and 2 fragments form (C) The vector is linearized as 1 fragment. (D) None of the above.

It not possible to infer mRNA sequence from a protein sequence because multiple codons code for the same amino acid. For instance, if a protein contains the amino acid Alanine, you wouldn't know whether the mRNA sequence that coded for that amino acid was GCU, GCA, GCG, or GCC. With DNA/mRNA however, cytosine in the mRNA strand is always the result of guanine in the DNA template strand.

Why is it not possible to infer mRNA sequence from a protein sequence while it is possible to infer mRNA sequence from DNA?

(C) Reverse Transcriptase cDNA is generated using the enzyme Reverse Transcriptase.

cDNA is generated using which of the following enzymes? (A) Ligase (B) Complimentary Synthase (C) Reverse Transcriptase (D) RNA Replicase