In a recombinant DNA cloning experiment, how can we determine whether DNA fragments of interest have been incorporated into plasmids and, once host cells are transformed, which cells contain recombinant DNA?

What steps make PCR a chain reaction that can produce millions of copies of a specific DNA molecule in a matter of hours without using host cells?

How has DNA-sequencing technology evolved in response to the emerging needs of genome scientists?

How can gene knockouts, transgenic animals, and gene editing techniques be used to explore gene function?

Write a short essay or sketch a diagram that provides an overview of how recombinant DNA techniques help geneticists study genes.

What roles do restriction enzymes, vectors, and host cells play in recombinant DNA studies? What role does DNA ligase perform in a DNA cloning experiment? How does the action of DNA ligase differ from the function of restriction enzymes?

The human insulin gene contains a number of sequences that are removed in the processing of the mRNA transcript. In spite of the fact that bacterial cells cannot excise these sequences from mRNA transcripts, explain how a gene like this can be cloned into a bacterial cell and produce insulin.

Although many cloning applications involve introducing recombinant DNA into bacterial host cells, many other cell types are also used as hosts for recombinant DNA. Why?

Using DNA sequencing on a cloned DNA segment, you recover the nucleotide sequence shown below. Does this segment contain a palindromic recognition sequence for a restriction enzyme? If so, what is the double-stranded sequence of the palindrome, and what enzyme would cut at this sequence?

CAGTATGGATCCCAT

Restriction sites are palindromic; that is, they read the same in the 5' to 3' direction on each strand of DNA. What is the advantage of having restriction sites organized this way?

List the advantages and disadvantages of using plasmids as cloning vectors. What advantages do BACs and YACs provide over plasmids as cloning vectors?

What are the advantages of using a restriction enzyme whose recognition site is relatively rare? When would you use such enzymes?

In the context of recombinant DNA technology, of what use is a probe?

If you performed a PCR experiment starting with only one copy of double-stranded DNA, approximately how many DNA molecules would be present in the reaction tube after 15 cycles of amplification?

In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges

(a) 92-26 °C

(b) 45-65 °C and

(c) 65-75 °C

We usually think of enzymes as being most active at around 37°C, yet in PCR the DNA polymerase is subjected to multiple exposures of relatively high temperatures and seems to function appropriately at 65–75°C. What is special about the DNA polymerase typically used in PCR?

Traditional Sanger sequencing has largely been replaced in recent years by next-generation and third-generation sequencing approaches. Describe advantages of these sequencing methods over first-generation Sanger sequencing.

How is fluorescent in situ hybridization (FISH) used to produce a spectral karyotype?

What is the difference between a knockout animal and a transgenic animal?

One complication of making a transgenic animal is that the transgene may integrate at random into the coding region, or the regulatory region, of an endogenous gene. What might be the consequences of such random integrations? How might this complicate genetic analysis of the transgene?

When disrupting a mouse gene by knockout, why is it desirable to breed mice until offspring homozygous (−/−) for the knockout target gene are obtained?

Gene targeting and gene editing are both techniques for removing or modifying a particular gene, each of which can produce the same ultimate goal. What is the main technical difference in how DNA is modified that differs between these approaches?

The CRISPR-Cas system has great potential but also raises many ethical issues about its potential applications because, theoretically, it can be used to edit any gene in the genome. What do you think are some of the concerns about the use of CRISPR-Cas on humans? Should CRISPR-Cas applications be limited for use on only certain human genes but not others? Explain your answers.

What is a single guide RNA, and what role does it play in CRISPR-Cas genome editing in eukaryotic cells?

What is the difference between nonhomologous end-joining (NHEJ) and homology-directed repair (HDR) in the context of genome editing?

What safety considerations must be taken before CRISPR-Cas is used to edit human embryos to cure disease?

Provide one example of a CRISPR-Cas application for biotechnology.