Molecular Biology Exam Review: DNA, RNA, PCR, Cloning, and CRISPR

What is the central dogma used in this course?

DNA is transcribed into RNA, and RNA is translated into protein.

What building blocks do DNA and RNA polymerases join together?

Nucleotides.

In what direction are new DNA and RNA strands synthesized?

5 prime to 3 prime.

In what direction is the template strand read?

3 prime to 5 prime.

To which end of a growing nucleic-acid strand is each new nucleotide added?

The 3 prime end.

Why can polymerases synthesize only in the 5 prime to 3 prime direction?

Because polymerase adds the incoming nucleotide to a free 3 prime hydroxyl group on the growing strand.

What does a primer provide that DNA polymerase needs?

A free 3 prime OH group.

Can DNA polymerase begin synthesis from nothing?

No, it needs a primer with a free 3 prime OH.

What does the incoming nucleotide contribute during strand extension?

Its 5 prime phosphate group.

What bond forms between nucleotides in the sugar-phosphate backbone?

A phosphodiester bond.

What type of bond holds the DNA backbone together?

Strong covalent phosphodiester bonds.

What type of bonds hold complementary DNA strands together?

Hydrogen bonds between complementary bases.

What gives DNA its negative charge?

The phosphate groups in the sugar-phosphate backbone.

Where is DNA's negative charge located?

Along the sugar-phosphate backbone.

What is the main chemical difference between DNA and RNA sugars?

RNA has a 2 prime OH on ribose; DNA lacks that oxygen at carbon 2 and contains deoxyribose.

What does the D in DNA remind you of?

Deoxy, meaning one oxygen is missing from the sugar compared with RNA.

Why is RNA less chemically stable than DNA?

Its 2 prime OH can participate in reactions that break the RNA backbone.

Why do ddNTPs stop DNA synthesis?

They lack a 3 prime OH, so the next nucleotide cannot be added.

In what technique are ddNTPs used?

Sanger sequencing.

What one rule connects polymerase activity, primers, ligase chemistry, and Sanger termination?

A free 3 prime OH is required to extend a nucleic-acid chain.

What is the first major step in DNA or RNA isolation?

Lyse the cells to release their contents.

After lysis, why is the sample centrifuged?

To separate heavy cellular debris from soluble molecules.

What are the two products after the first centrifugation?

A pellet at the bottom and a supernatant on top.

What is usually found in the first pellet?

Large organelles, membranes, proteins, and cellular debris.

Where are DNA and other soluble molecules after the first spin?

In the supernatant.

What extraction reagent pair is added to separate nucleic acids from proteins and lipids?

Phenol/chloroform.

How much phenol/chloroform should be added?

An equal volume to the aqueous sample.

After phenol/chloroform extraction, what are the major phases?

An upper aqueous phase, an interphase, and a lower organic phase.

Which phase contains DNA and RNA after phenol/chloroform extraction?

The aqueous phase, usually the top layer.

Why do DNA and RNA remain in the aqueous phase?

They are polar molecules and interact well with water.

What is mainly found in the organic phase and interphase?

Many proteins, lipids, membranes, and other cellular material.

Why must the interphase not be disturbed when collecting nucleic acids?

It contains contaminating proteins and debris that can reduce sample purity.

What does sodium acetate do during nucleic-acid precipitation?

Sodium ions shield or neutralize the negative phosphate charges.

Why does shielding the phosphate charges help DNA precipitate?

It reduces DNA-water interactions and allows DNA molecules to aggregate.

What does ethanol do during DNA or RNA precipitation?

It lowers nucleic-acid solubility so the nucleic acid comes out of solution as a pellet.

After adding salt and ethanol and centrifuging again, where is the DNA?

In the pellet.

What is used to wash a DNA pellet?

Usually 70 percent ethanol.

Why is 70 percent ethanol used instead of pure water?

It removes salts while keeping DNA precipitated.

What should be done after washing and drying the DNA pellet?

Resuspend it in water or an appropriate buffer.

Why can strong base ruin an RNA isolation?

NaOH rapidly hydrolyzes RNA because RNA has a reactive 2 prime OH.

If no RNA appears on a gel after NaOH was accidentally added, what likely happened?

The RNA was degraded.

What are RNases?

Enzymes that degrade RNA.

Why must RNA samples be protected from RNases?

RNases are common and can quickly destroy RNA.

What is the overall goal of nucleic-acid purification?

Keep DNA or RNA while removing debris, proteins, lipids, salts, nucleases, and other contaminants.

What is the most abundant type of RNA in cells?

Ribosomal RNA, or rRNA.

Which RNA carries coding information used to make protein?

Messenger RNA, or mRNA.

Is newly transcribed eukaryotic RNA immediately ready for translation?

No, it begins as pre-mRNA and must be processed.

What three major processing steps convert pre-mRNA into mature mRNA?

Addition of a 5 prime cap, removal of introns by splicing, and addition of a 3 prime poly-A tail.

What is found at the 5 prime end of mature eukaryotic mRNA?

A 5 prime cap.

What is found at the 3 prime end of mature eukaryotic mRNA?

A poly-A tail.

What happens to introns during mRNA processing?

They are removed, and exons are spliced together.

Why is mature mRNA important?

It is the RNA template that can be translated to produce protein.

What is a poly-A tail?

A long stretch of adenine nucleotides at the 3 prime end of many eukaryotic mRNAs.

Give two important functions of the poly-A tail.

It helps protect and stabilize mRNA, and it provides a known sequence for oligo(dT) binding.

What is oligo(dT)?

A short DNA primer or capture sequence made of many thymine bases.

Why does oligo(dT) bind mRNA?

Its thymine bases are complementary to the adenines in the poly-A tail.

How can oligo(dT) be used during mRNA purification?

Oligo(dT) attached to beads or a column captures polyadenylated eukaryotic mRNA.

How can oligo(dT) be used during cDNA synthesis?

It binds the poly-A tail and provides a free 3 prime OH for reverse transcriptase.

What is cDNA?

Complementary DNA, a DNA copy made from an RNA template.

Why convert RNA into cDNA?

DNA is more stable, and PCR enzymes amplify DNA rather than RNA.

What enzyme makes cDNA from RNA?

Reverse transcriptase.

Why does reverse transcriptase still need a primer?

It is a polymerase and needs a free 3 prime OH from which to extend.

What are the main ingredients for first-strand cDNA synthesis?

RNA template, a primer such as oligo(dT), dNTPs, reverse transcriptase, buffer, salts, and required ions such as magnesium.

What is the template during cDNA synthesis?

RNA, commonly mature mRNA.

What is the product of reverse transcription?

cDNA.

What information does a cDNA library represent?

The mature mRNAs expressed in the source cells at the time the RNA was collected.

Would a cDNA made from mature mRNA normally contain introns?

No. Mature mRNA has already had its introns removed.

Is poly-A/oligo(dT) enrichment mainly a eukaryotic mRNA strategy?

Yes. The standard long 3 prime poly-A tail framework applies mainly to eukaryotic mRNA.

What does PCR stand for?

Polymerase chain reaction.

Why is PCR performed?

To amplify a specific DNA sequence from a small starting amount.

What are the three repeating steps of PCR?

Denaturation, annealing, and extension.

What happens during denaturation?

Heat breaks hydrogen bonds and separates the two DNA strands.

What happens during annealing?

Primers bind to complementary sequences on the DNA templates.

What happens during extension?

DNA polymerase extends from each primer and synthesizes new DNA.

Which PCR step occurs at the highest temperature?

Denaturation.

Why is the annealing step performed at a lower temperature than denaturation?

Lower temperature allows primers to hybridize to complementary DNA sequences.

What determines which region of DNA is amplified in PCR?

The positions and sequences of the primers.

What DNA is amplified relative to the primers?

The DNA region lying between and downstream from the two primer-binding sites.

Why will a nearby gene not necessarily be amplified?

Without primers that flank that gene, polymerase will not specifically amplify it.

What does it mean if a PCR gel has no bands?

The target DNA did not amplify to a detectable level.

Give four possible causes of no PCR band.

Annealing temperature too high, extension time too short, a missing reagent, or degraded/absent template DNA.

Why can an annealing temperature that is too high cause no product?

Even perfectly matched primers may be unable to bind because the conditions are too stringent.

Why can an extension time that is too short cause no band for a long target?

Polymerase does not have enough time to finish copying the full-length product.

What rough extension-rate rule was emphasized for Taq polymerase?

About 1,000 base pairs per minute.

Approximately how long should extension be for a 3,000-bp product?

About 3 minutes.

What does it mean if a PCR gel shows too many bands?

Primers bound nonspecifically and multiple DNA regions were amplified.

How can you reduce nonspecific PCR bands by changing annealing temperature?

Raise the annealing temperature to increase stringency.

What does PCR stringency mean?

How strictly the primer must match the template in order to remain bound.

What happens when annealing temperature is too low?

Primers can bind imperfect or nonspecific sites, producing extra bands.

How can changing magnesium reduce nonspecific PCR products?

Reducing Mg2+ can decrease permissive polymerase activity and improve specificity.

Why is magnesium needed in PCR?

It is a required cofactor that helps DNA polymerase function.

What can happen if Mg2+ is too low?

Polymerase may work poorly and little or no product may form.

What can happen if Mg2+ is too high?

Nonspecific amplification may increase.

What is the ideal PCR amplification formula?

Final copies = starting copies multiplied by 2 to the power of the number of cycles.

What does 2 to the n represent in PCR?

The ideal fold amplification after n cycles.

If you begin with 10 DNA molecules and run 4 cycles, how many ideal copies form?

10 x 2^4 = 10 x 16 = 160 copies.

If you begin with 10 copies and run 5 cycles, how many ideal copies form?

10 x 2^5 = 10 x 32 = 320 copies.

Why is PCR amplification described as exponential?

Each product molecule can serve as a template in the next cycle, ideally doubling the amount every cycle.

What should a no-template control contain?

All PCR reagents except template DNA.

What does amplification in a no-template control suggest?

Contamination of the reagents or setup.