AP Biology Biotechnology

Gene cloning in prokaryotes

1) Isolate plasmid vector from bacterial cell and target gene (e.g. human insulin) from source DNA

2) Use the same restriction enzyme to cut both plasmid and target gene to create sticky ends

3) Use DNA ligase to covalently seal target gene into plasmid backbone, forming recombinant DNA

4) Introduce recombinant plasmid into prokaryotic cells via heat shock, making the cell membrane temporarily permeable to take up naked DNA

5) Grow bacteria on agar plates containing an antibiotic that matches an antibiotic-resistance gene on a plasmid (such as ampR) to make sure that only transformed bacteria with the plasmid survive

6) As bacteria reproduce, they clone the transformed recombinant plasmid, allowing scientists to isolate it and let it grow, then harvest the DNA (via centrifuge separation) or prompt the bacteria to transcribe and translate the gene into a protein product

Gene cloning in eukaryotes

1) Isolate mature mRNA from eukaryotic cell

2) Use reverse transcriptase to convert mRNA into single-stranded cDNA, then DNA polymerase to make the cDNA double-stranded

3) Use PCR to amplify select gene fragment

4) Cut cDNA and selected vector (e.g. plasmid, viral vector, yeast) with the same restriction enzyme to create sticky ends

5) Use ligase to seal the vector back up with cDNA gene of interest, ori, selectable marker (like antibiotic resistance), and an operon to regulate expression of the gene

6) Choose the host and insert cloned DNA through heat shock, viral transduction, etc.

7) Culture host cells on an antibiotic medium allowing only cells carrying the vector to survive, selecting for the resistant gene; screen to ensure the clones are without mutations

8) Grow host clones at a large scale to replicate recombinant DNA

PCR

1) Denaturation: high heat breaks down complementary base pairs; double-stranded DNA separates into two single strands

2) Annealing: mixture is cooled down; primers bind (anneal) hydrogen bonds to their complimentary sequences on the single-stranded DNA template

3) Elongation: temp is raised to optimate operating range for Taq polymerase, which synthesizes a new complementary DNA strand in a 5’ to 3’ direction, starting from primers

Each cycle doubles the number of target DNA molecules

Labeled nucleotides

Glowing tags are added to DNA like post-it notes to help scientists read the code

Dideoxyribose (ddDNA)

Modified five-carbon sugar that lacks a hydroxyl group (-OH) at both its 2’ and 3’ positions, possessing hydrogen atoms instead; used to form dideoxynucleotides in chain termination sequencing

Since it lacks a 3’ hydroxyl group, DNA polymerase cannot form a phosphodiester bond to attach to the next nucleotide, permanently halting DNA strand elongation

Chain termination sequencing

Determines the exact order of nucleotide bases in a DNA molecule

1) Heat double-stranded DNA to separate it into single-stranded DNA

2) Lower temperature and add a short, complimentary DNA primer to the 3’ end of the template strand to serve as the starting point for replication

3) Add DNA polymerase, standard deoxynucleotides, and a small amount of chain-terminating dideoxynucleotides (lacking a 3’ -OH group); polymerase synthesizes a new strand until a dideoxynucleotide is randomly incorporated, which stops farther expression

4) A mixture of single-stranded DNA fragments of various lengths is formed, each ending with a specific dideoxynucleotide

5) Separate resulting fragments by size using gel electrophoresis

6) Read separated fragments from smallest to largest (5’ to 3’) to deduce DNA sequence

cDNA

Complementary DNA: made by using reverse transcriptase to transcribe mRNA into artificial DNA

1) Isolate mRNA from cells

2) Primer binds to mRNA template

3) Reverse transcriptase synthesizes a single-stranded cDNA molecule complementary to the mRNA; original mRNA is degraded

4) DNA polymerase builds a second DNA strand complementary to the first cDNA strand, making double-stranded cDNA

5) DNA ligase helps seal fragments, creating a complete double-stranded molecule ready for cloning or PCR

RT-PCR

1) Extract RNA or mRNA from sample; remove contaminants

2) Treat RNA sample with double-strand specific DNase to degrade residual genomic DNA without damaging RNA template

3) Add primers so that reverse transcriptase can use RNA as a template to synthesize cDNA; heat the reaction to deactivate reverse transcriptase

4) Synthesized cDNA is transferred into a thermal cycler with Taq DNA polymerase and specifc primers; these steps repeat: A) Denaturation: high heat breaks down complementary base pairs; double-stranded DNA separates into two single strands; B) Annealing: mixture is cooled down; primers bind (anneal) hydrogen bonds to their complimentary sequences on the single-stranded DNA template; C) Elongation: temp is raised to optimate operating range for Taq polymerase, which synthesizes a new complementary DNA strand in a 5’ to 3’ direction, starting from primers

5) Quantify amplified product using techniques like gel electrophoresis

Can be used to identify RNA viruses like COVID, flu, HIV, and ebola

Microarray

Measures expression levels of thousands of genes at the same time

1) Isolate mRNA from both healthy and sick cells

2) Use reverse transcriptase to convert mRNA into more stable cDNA

3) Tag cDNA with fluorescent dyes to be able to distinguish between healthy and sick cells on the microarray; healthy is usually green while sick is usually red

4) Labeled cDNA samples are mixed and washed over the microarray slide; they hybridize with matching single-stranded probes on the chip

5) The chip is rinsed to wash away any unbound/non-specifically bonded cDNA

6) A laser scanner excites the dye and a computer records the color and intensity of light emitted from each micro-spot, determining which genes in the microarray are healthy or sick

Green=gene unregulated in healthy cells, red=unregulated in sick cells, yellow=equally expressed in healthy and sick cells, black=not expressed in either cell

Nucleic acid hybridization

The process of combining two complementary single-stranded nucleic acids to form a single, double-stranded molecule

1) Heat or chemical treatment denatures double-stranded DNA

2) Single-stranded, labeled DNA or RNA probes are added and allowed to anneal (bind) to complementary sequences

3) Unbound probes are washed away, and the presence of the target sequence is undetected

Probe

Short, single-stranded molecule of DNA or RNA used to locate and identify specific complementary nucleotide sequences

Its sequence is designed to match the target gene or mRNA of interest

When added to a sample, probe finds and hydrogen-bonds to its matching sequence

To track where it binds, probe is tagged with a marker, often a fluorescent dye

Gel electrophoresis

1) Isolate DNA and use restriction enzymes to cut it into specific fragments or amplify it using PCR; mix samples with buffer

2) Transfer DNA samples into wells; set up DNA ladder as a control (mixture of DNA fragments with known base pair lengths)

3) Turn on electricity; positive end near the wells and negative end on the opposite side; smaller fragments travel farther away from the wells since DNA has a negative charge

4) Stain gel with DNA-binding fluorescent dye and view the gel under UV light to analyze banding patterns and estimate fragment sizes

RFLP analysis

Restriction fragment length polymorphism analysis: identifies differences in homologous DNA sequences by measuring lengths of fragments produced by restriction enzymes

1) DNA is isolated from a sample

2) Restriction enzymes cut DNA at short recognition sequences

3) Resulted fragments undergo gel electrophoresis

4) Fragments are transferred onto a membrane and exposed to labeled, complementary DNA that binds to target sequence (southern blotting)

5) Pattern of bands is revealed using dyes to determine an individual’s specific genotype

Can be used to see paternity and genetic disease presence

How are DNA and RNA extracted from cells?

1) Detergents, enzymes, or mechanical disruption break open cell and nuclear membranes to release internal contents

2) Add agents to stop enzymes from degrading amino acids (RNA requires RNase-free conditions due to its fragility)

3) Cellular debris, proteins, and lipids are separated using organic solvents

4) Nucleic acids bind to silica columns or magnetic beads under specific salt/pH conditions; impurities are washed away via alcohol-based buffers

5) Purified DNA/RNA pellet is dissolved in a low-salt buffer or nuclease-free water

Genomic DNA vs cDNA

cDNA lacks introns since it is synthesized from spliced mRNA

Recombinant DNA

Artificially created DNA molecule formed by joining genetic material from multiple different sources or species that would not naturally combine in a genome

1) Target gene of interest is isolated from source organism

2) The same restriction enzyme cuts the DNA and carrier vector (e.g. plasmid) at specific sequences

3) Ligase links together the sticky ends left by the restriction enzyme, putting the gene of interest together with the ori, resistance gene, and operon, making recombinant DNA

4) Recombinant vector is introduced into a host cell like bacteria or yeast

5) As the host cell reproduces, it copies the recombinant DNA, allowing scientists to harvest resulting proteins or study the gene

May result in the production of human insulin from pigs, or the creation of pest resistant/herbicide tolerant GMOs

Cloning vector

Small, self-replicating DNA molecule (such as a bacterial plasmid or bacteriophages) used to carry foreign DNA into a host cell (like E. coli) so the gene can be copied or expressed

Made up of 1) Ori: Specific DNA sequence where replication usually starts, allowing the vector to replicate independently in the host bacterial cell

2) Recognition sequences are cut by restriction enzymes, creating sticky ends that match that of the gene of interest

3) Genes like antibiotic resistance (ampR) are added to the sticky end as a marker to show scientists whether the host cell took in the vector

4) Genes like lacZ help distinguish between empty and recombinant vectors

Bacteriophage

Specialized virus that infects, hijacks, and replicates inside bacterial cells

Are able to target specific bacteria without harming human cells

Lytic cycle

Fast, active process resulting in the destruction (lysis) of the host cell

1) Virus attaches to specific receptors on the host bacterial cell

2) Virus injects its genetic material (DNA or RNA) into the cell, leaving empty protein coat (capsid) outside

3) Viral genome hacks host cell’s cellular machinery and ribosomes to replicate viral DNA and synthesize viral proteins

4) Newly created viral components create new virions

5) Host cell bursts open (lyses), releasing the new virions to go infect neighboring cells, killing the host

Lysogenic cycle

Dormant, hidden process where virus replicates its genome without destroying its host

1) Phage attaches and injects its genetic material into the host cell

2) Viral DNA integrates itself into the host’s bacterium’s chromosome; at this stage, the integrated viral DNA is called a prophage

3) Each time the host cell divides normally, it copies its own DNA along with that of the viral prophage, passing it on to all daughter cells without alarming the host

4) In stressful conditions, the prophage can excise itself from the host chromosome and trigger the lytic cycle

Bacterial transformation

Process by which a bacterium uptakes foreign DNA (e.g. circular plasmid) from its environment and expresses the genes encoded on it

1) Cells (e.g. E. coli) are treated with cold CaCl2 to neutralize the negative charges of the DNA, increasing permeability of the membrane

2) Plasmid DNA (containing genes like ampicillin resistance or GFP) is added to the bacterial suspension

3) Tubes are rapidly moved from ice water to a warm bath, then back to ice; this creates pores in the bacterial membrane, allowing the plasmid to enter

4) Nutrient broth is added, and the bacteria incubate briefly so they can recover and begin expressing the antibiotic resistance gene

5) Culture is spread onto agar plates to screen for uptake

Southern blotting

1) Genomic DNA is cut into small pieces using restriction enzymes

2) Gel electrophoresis is used to separate DNA fragments by size on agarose gel

3) Gel is treated with alkaline solution to convert double-stranded DNA into single-stranded DNA

4) Transfer single-stranded DNA fragments from gel onto nylon/nitrocellulose membrane via capillary action

5) Introduce a labeled (fluorescent, radioactive, etc.) single-stranded DNA probe complementary to the target sequence

6) Wash away unbound probes and visualize bound probe using fluorescence/laser scanner

Stem cell

Unspecialized cells that are able to:

1) Divide continuously by mitosis while remaining unspecialized

2) Differentiate into specialized cell types

Totipotent cell

Unspecialized cell that can divide and differentiate into every cell type in an organism, including embryonic and placental tissues to support a complete, viable organism

Highest degree of cell potency

Pluripotent cell

Unspecialized stem cells that can self-renew indefinitely and differentiate into almost any cell type of the human body (specifically all cell types derived from the primary embryonic germ layers)

Operon

Cluster of prokaryotic genes under control of a single promoter and transcribed together into a single mRNA molecule

Made of 1) Promoter: sequence where RNA polymerase binds to begin transcription

2) Operator: DNA sequence acting as an on/off switch where a repressor protein binds

3) Structural genes: group of genes coding for related proteins or enzymes in a metabolic pathyway

4) Regulatory gene: usually located outside of operon; codes for repressor protein

Lac-Z operon (inducible)

Default state is off

1) Active repressor protein is bound to the operator, stopping RNA polymerase

2) Inducer (allolactose/lactose) binds to the repressor and changes its shape, causing it to fall off the operator so transcription can proceed

Usually breaks down molecules and is only turned on when the nutrient is present

Lactose present, glucose present: Low/negligible transcription

Lactose present, glucose absent: High transcription

Trp operon (repressible)

Default state is on

1) Repressor is inactive by default

2) Co-repressor (excess tryptophan) binds to the repressor, activating it so that it can attach to the operator and block RNA polymerase

Usually builds essential molecules (anabolic pathways) and turns off when end-product builds up in excess

DNA sequencing

1) Combine target single-stranded DNA template with primer, DNA polymerase, and regular deoxynucleotides; add a low concentration of fluorescently labeled chain-terminating dideoxynucleotides

2) Heat the mixture to separate double-stranded DNA into single strands (denaturation); cool mixture slightly so the primer can bind to the 3’ end of the template strand (annealing); raise the temperature for DNA polymerase to synthesize a new complementary DNA strand, adding ddNTPs until it randomly incorperates a fluorescent ddNTP (extension)

3) Mixture of labeled DNA fragments is loaded into a long glass tube containing a gel and subjected to an electric current; smaller fragments travel farther through the gel than large ones

4) As fragments pass a laser detector at the end of the tube, a sensor reads the fluorescent color of the ddNTP at the end of each fragment (shortest to longest); a computer translates these signals into the exact nucleotide sequence

Taq polymerase

Heat-stable DNA enzyme used to drive PCR; synthesizes new complementary DNA strands during the extension phase by adding free nucleotides in the 5’ to 3’ direction

Retroactive virus

RNA-based virus that transcribes its RNA genome into DNA using reverse transcriptase

1) Virus uses its outer envelope glycoproteins to attach to specific receptors on the host cell membrane; viral envelope fuses with host plasma membrane, releasing viral capsid into the cytoplasm, which breaks down to release two single-stranded viral RNA molecules and essential viral enzymes

2) Reverse transcriptase uses viral single-stranded RNA as a template to synthesize cDNA; reverse transcriptase degrades original viral RNA template and builds a matching second DNA strand, resulting in double-stranded viral DNA

3) Double-stranded viral DNA moves from cytoplasm into host cell’s nucleus; once integrated into host genome via integrase, the viral DNA is permanently part of the host genome and is called a provirus

4) RNA polymerase transcribes permanent provirus DNA into viral mRNA strands; host cell’s ribosomes translate this viral mRNA into long viral polyproteins

5) Viral RNA genomes and translated proteins travel to the cell membrane to package themselves into immature virus particles; immature virions push through the host cell’s plasma membrane, pinching off a piece of it to form their new lipid envelope; viral enzyme protease cuts the long, inactive polyproteins into functional, individual structural proteins and enzymes, turning the virion into a mature, infectious virus