Campbell Biology Chapter 20 Review: Biotechnology

20.1 Cutting, pasting and copying DNA

pp. 396–405

On the AP exam? Yes

Topic 6.8 tests what PCR and bacterial transformation do and how antibiotic selection picks out transformed cells; restriction enzymes and plasmids show up in lab questions. DNA libraries, BACs, probes and the blue-white screening details are background.

In the course: Topic 6.8 Biotechnology, Topic 6.1 DNA and RNA Structure, Topic 6.4 Translation (notes, videos and more questions)

Key points

Recombinant DNA is a lab-made molecule that joins DNA from two origins, such as a human gene and a bacterial plasmid. Biotechnology means using living things or their parts to make something useful, and genetic engineering is the branch that changes genes on purpose.
Restriction enzymes come from bacteria, which use them to chop up invading viral DNA. Each one recognizes its own short sequence, usually 4–8 base pairs long, and cuts both strands there. Many leave short single-stranded tails called sticky ends. Methyl groups on the bacterium's own DNA keep it from being cut.
Cut a plasmid and a piece of foreign DNA with the same enzyme, and their sticky ends match and can pair up. DNA ligase then seals the sugar-phosphate backbones with covalent bonds, giving a stable recombinant plasmid.
In bacterial transformation, cells take in the plasmid. The plasmid also carries an antibiotic resistance gene, so only transformed cells survive on that antibiotic. Each survivor grows into a colony of identical cells that all copy the plasmid and its gene (gene cloning). A second marker, like a lacZ gene that the insert breaks (white colonies instead of blue), can flag plasmids that picked up an insert.
Bacteria can't splice out introns, so scientists often clone cDNA instead: DNA copied from a cell's finished mRNA by reverse transcriptase, the enzyme retroviruses use. Proteins that need sugars or other groups added after translation are made in yeast or cultured animal cells, which have an ER and Golgi.
PCR copies one chosen stretch of DNA in a tube. Each cycle heats the DNA so the strands separate, cools it so two primers can bind at the two ends of the target, then warms it so a heat-stable polymerase (first found in a hot-spring bacterium) extends the primers. The target roughly doubles each cycle, so n cycles multiply it by about 2ⁿ.
A human gene can work in a bacterium at all because nearly every organism uses the same genetic code, which is strong evidence of common ancestry. Today PCR and made-to-order synthetic DNA have mostly replaced building and searching whole DNA libraries.

Key terms (15)

recombinant DNA: DNA made in the lab by joining pieces from two different sources, such as a human gene placed inside a bacterial plasmid.
genetic engineering: Changing an organism's genes on purpose, for example by adding a gene from another species.
plasmid: A small ring of DNA in many bacteria, separate from the main chromosome. Scientists use plasmids to carry genes into cells.
restriction enzyme: A bacterial enzyme that cuts DNA wherever it finds one particular short sequence, called its restriction site.
sticky end: A short single-stranded tail left by some restriction enzymes. It will pair up with a matching end made by the same enzyme.
DNA ligase: The enzyme that forms covalent bonds to seal breaks in a DNA backbone. In the lab it locks a pasted-in gene into place.
cloning vector: A carrier for moving a gene into a host cell, usually a plasmid. Once inside, it gets copied along with the gene it carries.
bacterial transformation: When a bacterium takes up DNA, such as a plasmid, from its surroundings. Labs use it to put new genes into bacteria.
gene cloning: Making many identical copies of a gene, usually by letting bacteria that carry it multiply.
complementary DNA (cDNA): DNA copied from mRNA by reverse transcriptase. It holds a gene's exons but none of its introns.
DNA library: A stored collection of bacterial clones that together carry many pieces of one organism's DNA (genomic library) or cDNA copies of its mRNAs (cDNA library).
nucleic acid probe: A short, labeled strand of DNA or RNA that pairs with a matching sequence, so you can find that sequence in a mixture.
polymerase chain reaction (PCR): A test-tube method that makes millions of copies of one chosen DNA segment through repeated cycles of heating and cooling.
primer: A short single strand of nucleic acid that pairs with a template and gives DNA polymerase a starting point. PCR uses two, one for each end of the target.
Taq polymerase: A DNA polymerase from a bacterium that lives in hot springs. It survives PCR's near-boiling steps, so it doesn't need replacing each cycle.

Check yourself: 20.1 Cutting, pasting and copying DNA

4 questions on 20.1 Cutting, pasting and copying DNA. Pick an answer to see if you got it, and why.

Question 1 of 4

A student cuts a plasmid and a fragment of human DNA with the same restriction enzyme, which leaves four-base sticky ends. She mixes the two but forgets to add DNA ligase before adding the mixture to bacteria. What is the most likely result?

Question 2 of 4

A plasmid carries two antibiotic resistance genes, one for ampicillin and one for tetracycline. A researcher inserts a foreign gene into a restriction site in the middle of the tetracycline resistance gene, transforms bacteria, and tests colonies on plates with each antibiotic. Which colonies most likely carry the plasmid with the foreign gene inserted?

Question 3 of 4

A researcher places two versions of a human enzyme gene into identical bacterial expression plasmids. Version 1 is copied straight from chromosomal DNA and is 15.2 kb long. Version 2 is made from the enzyme's mature mRNA with reverse transcriptase and is 1.9 kb long. Only bacteria carrying version 2 make active enzyme. Which explanation best fits these results?

Question 4 of 4

A human hormone works only when short sugar chains are attached to it. When its cDNA is expressed in E. coli, the protein has the correct amino acid sequence but is inactive. When the same cDNA is expressed in cultured hamster cells, the hormone is active. What best explains the difference?

0 of 4 answered

20.2 Sorting, reading and testing genes

pp. 405–412

On the AP exam? Yes

Topic 6.8 tests reading gels (DNA moves toward the positive end, and short pieces go farthest) and what sequencing tells you. Southern and Northern blots, microarrays and sequencing chemistry are background, and newer methods have replaced many of them.

In the course: Topic 6.8 Biotechnology, Topic 6.6 Gene Expression and Cell Specialization, Topic 6.2 DNA Replication, Topic 5.4 Non-Mendelian Genetics (notes, videos and more questions)

Key points

Gel electrophoresis sorts DNA by size. Phosphate groups make DNA negatively charged, so in an electric field every fragment heads for the positive electrode, and the gel's mesh lets short pieces slip through fastest. A ladder of fragments with known sizes, run in its own lane, lets you estimate each band's size.
Cutting DNA with a restriction enzyme and running it on a gel gives a band pattern. If a mutation creates or destroys a cut site, the pattern changes. Scientists once told alleles apart this way, by spotting restriction fragment length polymorphisms (RFLPs). Cut a linear piece in n places and you get n + 1 fragments; cut a circle n times and you get n.
Southern blotting moved DNA from a gel onto a membrane, then used a labeled single-stranded probe to light up only the bands carrying one gene. Northern blotting did the same with mRNA. Both rely on complementary base pairing, and PCR-based tests and sequencing have largely replaced them.
Dideoxy (Sanger) sequencing copies a template using normal nucleotides mixed with a few labeled ones that lack a 3′ –OH. Whenever a labeled one is added, that copy stops growing. Lining up the stopped copies by length reads the sequence base by base. Newer methods read millions of fragments at once, and a whole human genome now costs a tiny fraction of what it did in 2011.
To see where and when a gene is on, researchers look for its mRNA. RT-PCR turns mRNA into cDNA and then amplifies it, and in situ hybridization makes mRNA glow right inside a tissue. Microarrays once measured thousands of genes at a time; today RNA sequencing (RNA-seq) usually does that.
To learn what a gene does, scientists shut it off and see what changes. Older methods built knockout mice, and RNA interference uses double-stranded RNA to destroy a gene's mRNA. Since about 2012, CRISPR-Cas9 has let researchers edit or knock out almost any gene quickly.
Genome-wide association studies compare many people with and without a condition at SNPs, single-base sites that vary in the population. A SNP that's much more common in affected people usually just sits close to (is linked to) a gene that matters; it's a marker, not necessarily the cause.

Key terms (15)

gel electrophoresis: A way to separate DNA fragments by size: an electric current pulls them through a gel toward the positive end, and smaller pieces travel farther.
restriction fragment length polymorphism (RFLP): A difference between people's DNA that changes where a restriction enzyme cuts, so their fragments, and band patterns, come out different.
nucleic acid hybridization: Base pairing between two single strands from different sources, such as a labeled probe finding its matching DNA.
Southern blotting: An older method that transfers DNA bands from a gel to a membrane and uses a labeled probe to reveal just the bands carrying one sequence.
dideoxy (Sanger) sequencing: Sequencing that uses labeled nucleotides lacking a 3′ –OH, so each copy stops at a known base; sorting the copies by length reads the sequence.
next-generation sequencing: Newer, massively parallel methods that read millions of DNA fragments at once, making whole genomes fast and cheap to sequence.
RT-PCR: Reverse transcription followed by PCR. It copies mRNA into cDNA and then amplifies it, showing whether a gene is being expressed.
in situ hybridization: Using labeled probes on intact tissue or embryos to see exactly which cells contain a particular mRNA.
DNA microarray: A glass slide dotted with thousands of DNA sequences, used to measure how much mRNA from each gene a sample contains.
RNA sequencing (RNA-seq): Converting all the mRNA in a sample to cDNA and sequencing it, to measure which genes are on and how strongly.
gene knockout: An organism or cell whose copy of a particular gene has been disabled, so scientists can see what goes wrong without it.
RNA interference (RNAi): Silencing a gene with small or double-stranded RNAs that match its mRNA, causing that mRNA to be destroyed or blocked.
CRISPR-Cas9: A gene-editing tool adapted from a bacterial defense system. A guide RNA leads the Cas9 enzyme to a matching DNA site, where it cuts.
single nucleotide polymorphism (SNP): A single base position where people commonly differ, found in at least about 1% of a population.
genome-wide association study (GWAS): A study that scans many people's genomes for markers, like SNPs, that are more common in those with a particular condition.

Check yourself: 20.2 Sorting, reading and testing genes

4 questions on 20.2 Sorting, reading and testing genes. Pick an answer to see if you got it, and why.

Question 1 of 4

A sample containing DNA fragments of 300, 1,200 and 4,000 base pairs is loaded into a well at the negative end of an agarose gel, and the current is switched on. Which description of the result is correct?

Question 2 of 4

A 5,400 bp circular plasmid is cut with a restriction enzyme that has exactly two sites, at positions 900 and 3,300 (numbered around the circle). What should appear on the gel?

Question 3 of 4

In dideoxy (Sanger) sequencing, why does a growing DNA copy stop as soon as a dideoxynucleotide is added to its end?

Question 4 of 4

A researcher uses RT-PCR to test whether the gene for a flower-pigment enzyme is expressed in the petals, leaves and roots of one plant. She also tests each sample for the mRNA of a ribosomal protein gene that every living cell uses. The pigment gene gives a band only in petals; the ribosomal protein gene gives a band in all three tissues. What does the ribosomal protein result add to the experiment?

0 of 4 answered

20.3 Cloning organisms and stem cells

pp. 412–417

On the AP exam? Background

The exam won't ask about animal cloning or stem cell types by name, but this section is a strong example for Topic 6.6 (every cell has the same genes but uses different ones) and Topic 6.5 (epigenetic marks).

In the course: Topic 6.6 Gene Expression and Cell Specialization, Topic 6.5 Regulation of Gene Expression (notes, videos and more questions)

Key points

Organismal cloning makes a new individual with the same genes as a single parent. It answered an old question: do cells lose genes as they specialize? If one specialized cell can rebuild a whole organism, it must still carry the full genome. This idea is called genomic equivalence.
Many plant cells are totipotent: one mature plant cell, kept in a dish with the right nutrients and hormones, can rebuild an entire plant. Growers use this to copy plants with useful traits, and rooting a cutting is a simple form of cloning too.
Animals are cloned by nuclear transplantation (also called somatic cell nuclear transfer). The nucleus from a body cell goes into an egg whose own nucleus was removed, and the egg develops into an embryo. The first mammal cloned from an adult body cell was a sheep born in 1996, and in 2018 researchers reported the first monkeys cloned this way.
A clone's chromosomes match the nucleus donor, but its mitochondria come from the egg. Clones aren't perfect copies, because environment and chance shape development. Most cloned embryos fail or have health problems, largely because the donor nucleus keeps epigenetic marks, like DNA methylation, from its old job.
Stem cells can divide to make more stem cells and can also turn into specialized cells. Embryonic stem (ES) cells come from an early embryo (the blastocyst) and are pluripotent: they can become any body cell type. Adult stem cells, such as those in bone marrow, make only a limited set of cell types.
Reproductive cloning aims to produce a new animal. Therapeutic cloning would create an embryo only to collect ES cells that match a patient. Because using human embryos is controversial, it was a big step when, in 2006–2007, adding genes for a few transcription factors turned mouse and then human skin cells into induced pluripotent stem (iPS) cells. That work won a share of the 2012 Nobel Prize.
iPS cells can be made from a patient's own cells, so tissue grown from them should match the patient. They're widely used to study diseases in a dish, and treatments based on them are now being tested in clinical trials.

Key terms (14)

organismal cloning: Producing a whole new organism that's genetically identical to one parent, starting from a single cell or nucleus.
genomic equivalence: The idea that nearly all of an organism's cells carry the same complete set of genes, even after they specialize.
totipotent: Able to give rise to every cell type of an organism, including, in mammals, the tissues that support the embryo. A fertilized egg is totipotent.
pluripotent: Able to become any type of body cell, but not a whole organism on its own. Embryonic stem cells are pluripotent.
nuclear transplantation: Putting the nucleus of one cell into an egg whose own nucleus was removed. It's the main way animals are cloned.
enucleated egg: An egg cell whose nucleus has been taken out, ready to receive a donor nucleus.
reproductive cloning: Cloning done to produce a new individual animal.
therapeutic cloning: Making a cloned embryo only to harvest stem cells for medical treatment, not to produce a new individual.
stem cell: An unspecialized cell that can keep dividing to renew itself and can also turn into one or more kinds of specialized cell.
embryonic stem (ES) cell: A pluripotent stem cell taken from the inner part of an early embryo. It can become any body cell type.
adult stem cell: A stem cell in a grown body, such as in bone marrow or skin, that replaces cells in its own tissue and makes only a few cell types.
blastocyst: A hollow ball of cells that a mammal embryo forms a few days after fertilization. ES cells are taken from its inner cluster of cells.
induced pluripotent stem (iPS) cell: A specialized cell reprogrammed in the lab, by adding a few key transcription factor genes, to act like an embryonic stem cell.
regenerative medicine: Medicine that aims to repair or replace damaged tissues, for example with cells grown from stem cells.

Check yourself: 20.3 Cloning organisms and stem cells

4 questions on 20.3 Cloning organisms and stem cells. Pick an answer to see if you got it, and why.

Question 1 of 4

Consider three human cells: (1) a fertilized egg, (2) a cell from the inner cell mass of a five-day-old embryo, and (3) a bone marrow stem cell that makes red blood cells, white blood cells and platelets. Which list orders them from the greatest to the least developmental potential?

Question 2 of 4

A nursery grows 5,000 cassava plants by culturing single cells, all taken from one plant that resists a common leaf virus. A few years later a new fungal disease reaches the region. Compared with a field planted from the seeds of many different parents, what is most likely true of the cloned field?

Question 3 of 4

Question 4 of 4

Scientists turn adult skin cells into stem-like cells by adding extra copies of just four genes, each coding for a transcription factor. Why can so few genes cause such a big change?

0 of 4 answered

20.4 Biotechnology in medicine, forensics and farming

pp. 417–423

On the AP exam? Yes

Topic 6.8 tests using DNA results, such as matching samples on a gel, and knowing that a GMO carries a gene added on purpose. Gene therapy, 'pharm' animals and the rules for GM crops are background, and the book's medical examples are out of date.

In the course: Topic 6.8 Biotechnology, Topic 7.3 Artificial Selection, Topic 6.4 Translation (notes, videos and more questions)

Key points

Diagnosis: PCR with primers that match a gene can show whether someone carries a disease allele, even before symptoms or before birth. RT-PCR detects RNA viruses, and it was the standard lab test for COVID-19. SNP-based tests report risk, which is a correlation, not a sure prediction.
Gene therapy adds a working allele to a patient's own cells, often using a disabled virus as the carrier. Early trials around 2000 helped some children with an inherited immune disorder, but a few developed leukemia because the virus landed next to a gene that controls cell growth. Since 2017, several gene therapies have been approved, and in 2023 the first CRISPR-based treatment was approved for sickle cell disease.
Somatic gene therapy changes only body cells, so it isn't passed on to children. Changing eggs, sperm or embryos (germline editing) would be inherited. It's banned or tightly restricted in most countries, and a 2018 case of gene-edited babies in China was widely condemned.
Bacteria, yeast and cultured cells carrying human genes make medicines like insulin and growth hormone. Transgenic animals can make a human protein in their milk or eggs, where it's easier to collect.
A genetic profile compares DNA markers that vary a lot between people, mainly short tandem repeats (STRs): short sequences repeated a different number of times in different people. PCR copies each STR and electrophoresis measures its length. Since 2017 the US national database has used 20 core STRs. One mismatch excludes a person, while a full match is extremely unlikely by chance. The same idea settles paternity and identifies remains.
Engineered microbes can help clean up pollution or recover metals. GM crops carry genes for traits like insect resistance or herbicide tolerance. Unlike selective breeding, genetic engineering moves specific genes directly, even between species.
Concerns include crop genes spreading to wild relatives through pollen, pests evolving resistance, possible new allergens, and the privacy of genetic information. In the US, foods with detectable engineered genetic material have needed a 'bioengineered' disclosure since 2022.

Key terms (11)

gene therapy: Treating a disorder by adding a working copy of a gene to a patient's cells, or by editing the faulty one.
viral vector: A virus with its disease-causing genes removed, used to carry a therapeutic gene into human cells.
germline editing: Changing the DNA of eggs, sperm or early embryos, so the change would be passed on to future generations.
transgenic organism: An organism carrying a gene from another species, put there on purpose. That added gene is called a transgene.
genetically modified organism (GMO): Any organism whose DNA has been changed with genetic engineering, such as a crop given an insect-resistance gene.
genetic profile: A person's set of results at several highly variable DNA markers. It's nearly unique to each person, except identical twins.
short tandem repeat (STR): A stretch of DNA where a 2–6 base sequence repeats back to back. The number of repeats varies a lot between people.
personalized medicine: Choosing prevention or treatment based on a person's own genetic information.
Ti plasmid: A plasmid carried by Agrobacterium, a bacterium that lives in soil. It can insert part of its DNA into plant chromosomes. Scientists use disarmed versions to add genes to crops.
bioremediation: Using living things, often microbes, to break down or remove pollutants from soil or water.
Bt crop: A crop given a gene from the bacterium Bacillus thuringiensis so that it makes a protein toxic to certain insect pests.

Check yourself: 20.4 Biotechnology in medicine, forensics and farming

4 questions on 20.4 Biotechnology in medicine, forensics and farming. Pick an answer to see if you got it, and why.

Question 1 of 4

Wildlife officers find the carcass of a deer shot out of season. They test three STR markers in the carcass and in venison taken from three hunters' freezers (numbers are repeat counts for the two alleles at each marker). Sample | Marker 1 | Marker 2 | Marker 3 Carcass | 11, 14 | 7, 9 | 18, 18 Freezer 1 | 11, 14 | 7, 10 | 18, 18 Freezer 2 | 11, 14 | 7, 9 | 18, 18 Freezer 3 | 12, 14 | 7, 9 | 18, 20 Which conclusion do these data support?

Question 2 of 4Calculator allowed

At three STR markers, the genotypes in a DNA profile occur in 1 in 20, 1 in 10 and 1 in 50 people in the population. The markers are on different chromosomes and are inherited independently. About how likely is it that a random, unrelated person has the same genotypes at all three markers?

Question 3 of 4

A woman with an inherited blood disorder has some of her blood stem cells removed. A viral vector gives them a working copy of the faulty gene, and the cells are returned to her bone marrow. The therapy cures her. If she later has children, what is most likely true?

Question 4 of 4

Field mustard, a weedy wild plant, can cross-pollinate with canola. Researchers collect seeds from wild field mustard growing at different distances from a field of canola engineered to tolerate a herbicide. They grow all the seedlings in the same greenhouse and spray them with that herbicide (invented data). Distance from canola field | Seedlings surviving the spray 10 m | 7.5% 100 m | 2.1% 1 km | 0.3% 10 km | 0% Which explanation best fits these data?

0 of 4 answered