Theory of Recombinant DNA Techniques

When choosing primers for PCR

The 3' ends of the two primers should point towards each other following annealing to the DNA template strands

Which of the following components is required for DNA sequencing using the Sanger method?

dideoxynucleotide triphosphates for chain termination

The number of bases produced by different sequencing reactions varies widely. This is represented by which of the following approximate number of bases for each reaction with: i) Sanger dideoxy, ii) next generation (e.g. pyrosequencing), and iii) nanopore

i) 1000, ii) 50 to 100, iii) tens of thousands

At the current time, recombinant DNA research in the United States:

is allowed, but is subject to rules originally laid out at the Asilomar conference (1975), and currently administered by the National Institutes of Health (NIH

After sequencing a new genome, you search for open reading frames, and then compare the predicted protein sequences with all other known protein sequences using BLASTp. What will this tell you about the probable function of proteins encoded by the open reading frames?

it will suggest the function of many proteins that are similar to proteins of known function. C. It won't tell you the probable function of a significant percentage of proteins, since some of these are homologous to proteins with no known function, or have no known homologs anywhere.

In PCR reactions, the single-stranded DNA primers:

may contain extra sequences at the 5' that are not complementary to the template DNA

To use a PCR reaction to quantify the levels of a specific RNA, a researcher must

measure the total amount of DNA produced at the end of the PCR reaction determine the round in the qRT PCR at which the threshold of detection is reached

DNA ligase, an important component of cloning protocols

requires a 5' phosphate in close proximity to a 3' hydroxyl group for proper enzymatic function

The CRISPR Cas9 protein is a nuclease that recognizes

specific DNA (non PAM) sequences indirectly, through guide RNA base pairing with target DNA

One of the steps commonly used in DNA purifications is the addition of a high concentration of ethanol, along with salt. What is function of this step?

the ethanol/salt precipitates DNA, allowing it to be concentrated to a smaller volume

One of your classmates tells you: "I've figured out what this new open reading frame encodes — a BLAST search indicates it is most similar to the gene for pyruvate kinase!" You want to share in the enthusiasm, but first you want proof. So you ask what the E-value is for the alignment with pyruvate kinase, and your colleague replies "the E-value is 10-60!" What's the most appropriate response?

"Yes, with an E-value that low, it's quite likely to have pyruvate kinase activity"

When completing genome sequences, contiguous sequences, or contigs are important intermediates. Which of the following is true about contigs?

A contig represents a DNA sequence assembled from smaller sequencing reads, and has no gaps

he genome of the 2019 novel coronavirus (nCoV-2) had its complete genome sequenced very soon after the first COVID-19 cases were reported in China. Which of the following is true regarding the viral genome sequencing approach?The virus was completely sequenced using a Sanger dideoxy sequencing approach.A next generation approach was used, that utilized amplified DNA nanoballs and a ligation-driven sequencing technique.Since the viral genome is RNA, it could only be sequenced using a nanopore device.Since the viral genome is RNA, it had to be converted to cDNA with reverse transcriptase prior to being sequenced.Both b. and d. are correct.

A next generation approach was used, that utilized amplified DNA nanoballs and a ligation-driven sequencing technique Since the viral genome is RNA, it had to be converted to cDNA with reverse transcriptase prior to being sequenced.

Genome annotation occurs following completion of genome sequencing and assembly. List two different kinds of genome annotations, and the kind of biological information each annotation provides about the sequence

After a genome has been sequenced using either the Sanger or next generation methods, the genome can be annotated. To know which parts are coding or non coding the genome can be annotated using different methods.1)Protein Sequence -blastp, compares proteins in the sequence to compare if function might be similar2) DNA sequence- blastn, compares nucleotide in the sequence to determine is lineage might be similar, and to look for similar functions

The Asilomar Conference on Recombinant DNA (rDNA) in 1975 led to recommendations for rDNA research, including general safety recommendations that include:

B. All personnel working with rDNA must be trained in best safety and containment practices

How is PCR product accumulation usually detected during quantitative, real-time PCR (Q-RT PCR) assays?

Dyes (such as SYBR Green) bind to the PCR product, and if no quencher is nearby, will give an increase in fluorescence

What makes RNA isolation and purification more difficult than DNA isolation and purification?

Enzymes called RNases act very quickly to break down RNA, and inactivation/removal of RNases requires stringent precautions

You perform a BLAST search to try and figure out the potential function of a new open reading frame from your lab's DNA sequencing project. The best hit in the search is to a protein (encoding beta-galactosidase) that has a long region with 30% identity to the potential protein encoded by the open reading frame. What can you say about your open reading frame's protein product?

It could encode a protein with beta-galactosidase activity, but it would be good to purify the protein and test its activity before making a definitive conclusion.

The annealing temperature for a PCR reaction

can be varied to improve specificity of base pairing by DNA primers.

Which of the following scientific discoveries caused a moratorium on recombinant DNA research and led to the Asilomar conference on recombinant DNA, held in 1975?

Paul Berg and co-workers created the first artificial recombinant DNA molecule

How does the process of phenol extraction remove proteins from aqueous solutions?

Phenol is an organic solvent that causes protein unfolding and aggregation, leading to insolubility in aqueous solutions. Phenol does not affect solubility of nucleic acids, so DNA and RNA are not precipitated, and can be recovered in the aqueous phase of the extraction

Whole genome amplification (WGA) with phi29 DNA polymerase differs from the standard polymerase chain reaction (PCR) in which important way

Priming for WGA is done using random 6 nucleotide-long primers (hexamers)

Pyrosequencing is an example of a "next generation" DNA sequencing technique that involves measurement of changes in pyrophosphate levels. Why is it so useful in reducing the costs associated with the traditional Sanger sequencing approach?

Pyrosequencing allows many sequencing reactions (> 100,000) to be done in parallel.

You wish to add single-stranded polynucleotide tails to the 3' ends of a DNA fragment in a template-independent manner, to assist in subsequent cloning steps. Which of the following enzymes will perform this reaction?

Terminal transferase

With nanopore based sequencing, DNA is passed through a pore and the sequence is read in the process. If this method is perfected, why is it likely to be revolutionary

The method can theoretically read the sequences of very long DNA strands (tens to hundreds of thousands of bases in length) in a single reaction

What are features of the SARS-nCoV-2 genome that were revealed by its sequence?

The genome consists of RNA that is 29,829 bases in length.

When large genomes are sequenced, which of the following is true?

The genomes are fragmented, the DNA fragments are sequenced by any number of methods, and the sequences assembled to provide the original complete genome.

Type II restriction endonucleases (also called restriction enzymes)

bind to double stranded DNA at short, specific DNA sequences, and cut both DNA strands.

Why are dideoxynucleotide triphosphates useful in DNA sequencing?

They are missing a hydroxyl group at the 3' position of the ribose sugar. Because of the lack of a hydroxyl group, they cannot be extended by DNA polymerase. After DNA polymerase adds a ddNTP, the chain stops, and terminated chains can be separated by size on a gel or in a capillary. ddNTPs can be labelled with fluorescence so the identity of the ddNTP can be tracked in terminated chains.

In February 2020, a research paper was published to report the genome sequence of SARS nCoV2, the causative agent of COVID-19. Viruses from 9 different patients were sequenced, and yielded 8 complete genome sequences. Were the genomes identical to each other, or were there changes among the sequences?

Three of the genomes had identical sequence, while in the other 5 genomes there were 9 nucleotide positions with mutations

Some protocols for plasmid DNA purification utilize a silica matrix (usually in the form of a column). What is the purpose of the silica matrix

Under high salt conditions, DNA sticks to the silica while proteins and RNA pass through and are washed away

You are attempting to determine the function of a protein encoded by an open reading frame found in the sequenced genome of the plant Zea mays. You start by doing a BLAST search at NCBI using the predicted protein sequence. What can this approach tell you, and how does it compare to experiments using standard methods of genetics and biochemistry? Describe at least one thing you should you be careful of when interpreting BLAST results to describe function.

Using blastp can help you determine if the proteins in the sequences are similar, and therefore if the function of the sequence is similar.It can be very fast, and compares many different sequences at once instead of with traditional methods when you would have to compare one at a time.However, with blastp you have to be careful because similar structure does not always equal similar function, and further evaluation is needed before you can make that assumption.

What is the names of Dr. Bartletts rabbits?

Yes! Good answer, and remembering that she's a lop (Holland mini-lop, to be specific). The other is Momo

A general rule for genome sequencing projects is

the longer the DNA sequence yield per reaction, the easier it is to assemble the complete genome sequence.

You've just finished sequencing a eukaryotic genome using the Sanger dideoxy method, with a combination shotgun and ordered clone sequencing approach. Which of the following are good indicators that you can stop sequencing and fragment assembly, and are ready to begin annotation?

your genome size is ~ 1.5 billion base pairs, and you've collected ~15 billion base pairs of sequence information [10-fold (10X) coverage]. and Your scaffold N50 length is at least the median size of a eukaryotic gene including introns and eons.