AP Biology 6.7-6.8: Mutations and Biotechnology

Biotechnology

The use of biological organisms, cells, or molecules to develop useful products or processes. Major applications include medicine, agriculture, industrial fermentation, biofuels, and environmental cleanup.

Nucleic acid extraction

The isolation of DNA or RNA from cells for analysis or manipulation. Typical steps include cell lysis, degradation or removal of unwanted macromolecules, and precipitation or purification of the nucleic acid.

What roles do lysis buffer, protease, RNase, and alcohol play in DNA extraction?

A detergent-containing lysis buffer disrupts cell and nuclear membranes. Protease degrades proteins, RNase removes RNA, and alcohol precipitates DNA so it can be collected.

Why is RNA generally more difficult to isolate and analyze than DNA?

RNA is less chemically stable and is readily degraded by ubiquitous RNase enzymes. RNA extraction therefore requires especially careful use of buffers and RNase-inactivation methods.

Gel electrophoresis

A technique that separates charged molecules as they move through a porous gel in an electric field. DNA and RNA fragments are separated primarily by size.

Why do nucleic acids migrate in an electric field during gel electrophoresis?

Phosphate groups give DNA and RNA an overall negative charge at neutral or basic pH. Therefore, they are attracted toward the positive electrode.

How does fragment size affect DNA movement through an electrophoresis gel?

Smaller DNA fragments pass through the gel pores more easily and migrate faster and farther than larger fragments. Thus, smaller fragments are found closer to the positive electrode.

How can a DNA ladder be used in gel electrophoresis?

A DNA ladder contains fragments of known sizes and is run beside the unknown samples. The distances traveled by sample bands can be compared with the ladder to estimate their sizes.

What electrophoresis patterns are expected for uncut genomic DNA versus a mixture of genomic DNA fragments?

Uncut genomic DNA is usually too large to move far and appears as a large band near the wells. Fragments with many different sizes produce a continuous smear.

Denaturation and reannealing of DNA

Heating DNA disrupts the hydrogen bonds between complementary strands, causing denaturation. Cooling allows complementary strands to reanneal or hybridize.

Polymerase chain reaction (PCR)

PCR is an in vitro method for exponentially amplifying a specific DNA region. It uses repeated cycles of denaturation, primer annealing, and DNA synthesis by a thermostable DNA polymerase.

What components are required for a PCR reaction, and what is the function of each?

PCR requires template DNA, two primers, deoxynucleotides, a buffer with appropriate ions, and a thermostable DNA polymerase. The primers define the region amplified, and the polymerase extends them using the deoxynucleotides.

What determines which DNA sequence is amplified during PCR?

The two primers are designed to be complementary to sequences flanking the target region. DNA between the primer-binding sites is selectively amplified.

Why is Taq polymerase useful in PCR?

Taq polymerase comes from the thermophilic bacterium Thermus aquaticus and remains functional after the high-temperature denaturation step. This eliminates the need to add new polymerase after every cycle.

How does PCR achieve exponential amplification?

Ideally, each cycle doubles the amount of target DNA, so after $n$ cycles the amount is approximately proportional to $2^n$ times the starting amount. In practice, amplification eventually slows as reagents become limiting.

Reverse-transcription PCR (RT-PCR)

RT-PCR begins with an RNA template, usually mRNA. Reverse transcriptase first produces complementary DNA (cDNA), which is then amplified using standard PCR.

Why is cDNA useful for studying gene expression?

cDNA is made from mRNA, so it represents genes that were actively transcribed in the sampled cells. It can be amplified and analyzed more readily than unstable RNA.

DNA or RNA probe

A short, labeled nucleic acid sequence designed to hybridize with a complementary target sequence. Fluorescent or radioactive labels allow the target to be detected.

Southern blotting versus Northern blotting

Southern blotting transfers separated DNA fragments from a gel to a membrane and uses probes to detect a particular DNA sequence. Northern blotting applies the analogous procedure to RNA and is used to examine gene expression.

What is the purpose of transferring nucleic acids from a gel to a membrane during blotting?

The transfer immobilizes the separated DNA or RNA fragments on a membrane where they can be exposed to a labeled complementary probe. A signal indicates that the target sequence is present.

Molecular cloning

The production of many copies of a selected DNA fragment, often by inserting it into a plasmid and propagating the plasmid in bacteria. It differs from reproductive cloning, which aims to produce an entire genetically similar organism.

Plasmid vector

A small circular DNA molecule that replicates independently of bacterial chromosomal DNA. Engineered plasmids can carry foreign DNA into host cells and often include selectable or expression-related sequences.

Multiple cloning site (MCS)

A short engineered region of a plasmid containing recognition sites for several restriction enzymes. It provides convenient locations for inserting foreign DNA.

Restriction endonuclease

An enzyme that recognizes a specific DNA sequence and cuts the DNA at predictable locations. Bacteria naturally use these enzymes as a defense against foreign DNA.

Sticky ends

Short single-stranded DNA overhangs produced when many restriction enzymes make staggered cuts. Complementary sticky ends can temporarily base-pair, helping DNA fragments from different sources align.

DNA ligase

An enzyme that permanently joins aligned DNA fragments by forming phosphodiester bonds in the sugar-phosphate backbone. It is used to seal an inserted DNA fragment into a cut plasmid.

How can the same restriction enzyme be used to insert a foreign DNA fragment into a plasmid?

Cutting both the plasmid and the foreign DNA with the same restriction enzyme creates compatible ends. The fragments anneal through complementary bases, and DNA ligase seals the backbones.

Recombinant DNA and recombinant protein

Recombinant DNA is an artificially constructed DNA molecule containing sequences from different sources. A recombinant protein is produced when a host cell expresses a gene carried by that recombinant DNA.

Why might a recombinant plasmid replicate in a host cell but fail to produce the desired protein?

Replication and gene expression require different regulatory elements. The plasmid or host may lack a compatible promoter, regulatory sequence, or cellular machinery needed for transcription and translation.

What distinguishes cellular cloning from reproductive cloning?

Cellular cloning is the production of genetically similar cells, such as bacterial or yeast cells formed by asexual division. Reproductive cloning produces an entire multicellular organism genetically similar to a donor.

Genetic engineering

The deliberate alteration of an organism's genotype using recombinant DNA methods to produce a desired trait. Gene insertion, mutation, and deletion are possible approaches.

Genetically modified organism (GMO) versus transgenic organism

A GMO has had its genetic material altered using biotechnology. A transgenic organism is a GMO that contains DNA introduced from a different species.

Gene targeting and reverse genetics

Gene targeting uses recombinant DNA methods to alter, disrupt, or delete a particular gene. It is a reverse-genetics strategy because researchers begin with a DNA sequence change and then observe its phenotypic effect.

Genetic diagnosis

The use of genetic testing to identify disease-associated mutations or other inherited risk factors before selecting treatment or screening strategies. Testing may be performed on affected individuals, relatives, fetuses, or embryos produced by in vitro fertilization.

Gene therapy

A treatment strategy that introduces, replaces, or edits genetic material to address a disease caused by a defective gene. A viral vector may deliver a functional gene, although newer approaches can correct the mutation at its original genomic location.

How do recombinant vaccines stimulate immunity?

A gene encoding a pathogen antigen is cloned and expressed to produce large quantities of the antigen. Introducing the antigen stimulates a primary immune response and formation of immune memory without requiring the entire pathogen.

How has recombinant DNA technology changed production of human insulin?

The human insulin gene can be inserted into a bacterial vector and expressed in E. coli. This provides a scalable source of human insulin and avoids some immune reactions associated with insulin from nonhuman animals.

Why are some therapeutic proteins produced in transgenic animals rather than bacteria?

Some proteins require eukaryotic folding or post-translational processing that bacterial cells cannot perform correctly. Transgenic sheep, goats, chickens, or mice can produce these proteins in milk or eggs.

Somatic cell nuclear transfer (SCNT)

SCNT removes the haploid nucleus from an egg and replaces it with a diploid nucleus from a donor somatic cell. The reconstructed egg can begin embryonic development and may be used for reproductive or therapeutic cloning.

Why can an enucleated egg with a donor somatic-cell nucleus develop into an embryo?

The donor nucleus supplies diploid genetic information, while the egg cytoplasm supplies molecules and organelles needed for early development. The resulting embryo is genetically similar in nuclear DNA to the donor.

Parthenogenesis

A form of asexual reproduction in which an embryo develops from an unfertilized egg. The offspring may be haploid or arise through mechanisms that restore diploidy, depending on the species.

Reproductive cloning versus therapeutic cloning

Reproductive cloning aims to produce an entire organism genetically similar to a donor. Therapeutic cloning aims to generate genetically compatible stem cells or tissues for research or treatment rather than produce a new organism.

Applications of transgenic plants

Plant genetic engineering can produce disease, pest, herbicide, or environmental-stress resistance, as well as improved nutrition and shelf life. Ecological testing is important because introduced genes may spread to other populations.

How is Agrobacterium tumefaciens used to genetically transform plants?

Researchers modify the bacterium's Ti plasmid by removing tumor-causing genes and inserting a desired gene. The bacterium naturally transfers part of this plasmid into the plant genome, where the introduced DNA can be expressed.

Bt crops

Bt crops contain genes from Bacillus thuringiensis that encode insecticidal crystal proteins. In susceptible insects, ingestion and activation of the protein in the intestine stop feeding and eventually cause death.

Why is Bt considered a selective biological insecticide?

The toxin must be eaten and activated in the digestive tract of susceptible insects, making it generally less harmful to humans and other mammals. Its use can reduce dependence on externally applied chemical pesticides, although ecological monitoring remains necessary.

Antisense RNA technology in crop engineering

Antisense RNA is designed to be complementary to a target mRNA and reduce its translation or stability. In the Flavr Savr tomato, this approach slowed softening and extended shelf life.

Mutation

A heritable change in the nucleotide sequence of DNA. Mutations can arise spontaneously from replication or repair errors or be induced by mutagens such as radiation and certain chemicals.

Point mutation

A small-scale mutation involving a change in a single nucleotide or base pair. A point mutation may be silent, missense, or nonsense depending on how it changes the encoded protein.

Frameshift mutation

An insertion or deletion of nucleotides that is not a multiple of three, shifting the reading frame of the codons downstream. Frameshifts often alter many amino acids and can create a premature stop codon.

Silent mutation

A nucleotide substitution that changes a codon but not the encoded amino acid because the genetic code is redundant. It may have little or no effect on protein function.

Missense mutation

A nucleotide substitution that changes a codon so that a different amino acid is incorporated into the protein. Its effect can range from negligible to severe depending on the amino acid and protein location.

Nonsense mutation

A nucleotide substitution that converts an amino-acid codon into a stop codon. Translation ends prematurely, usually producing a shortened and nonfunctional protein.

How can a mutation affect phenotype?

A mutation may have a neutral, harmful, or beneficial effect. Its consequence depends on its location, whether it changes gene expression or protein sequence, and whether the altered protein remains functional.

Somatic versus germline mutation

A somatic mutation occurs in a non-reproductive cell and is generally not transmitted to offspring, although it may affect the individual or contribute to cancer. A germline mutation occurs in cells that produce gametes and can be inherited.

CRISPR-Cas9

A genome-editing system in which a guide RNA directs the Cas9 nuclease to a complementary DNA sequence. Cas9 makes a targeted double-strand break, which the cell repairs to disrupt, remove, or alter the gene.

How can CRISPR-Cas9 be used to alter a gene?

Researchers design a guide RNA complementary to the target sequence. Cas9 cuts at that location, and cellular repair can introduce insertions or deletions that disable the gene or use a supplied DNA template to make a precise change.