9 Biotechnology

biotechnology

The use of microbiological (microbes and microorganisms) and biochemical techniques (enzymes) to solve practical problems and produce product

→ creates protein sources that are reliably available and less expensive than traditional sources

→ genetic engineering of organisms (GMOs) using recombinant DNA techniques: methods used to manipulate DNA and genetically alter organisms

Genetically modified organisms

GMOs. Organisms that have been genetically engineered to confer useful traits

Restriction enzymes

Enzymes naturally occurring in organisms that have the ability to recognize palindromes: specific sequences in the DNA that are mirror inversions of each other, and cut them

→ palindromes are consistent throughout all of the sequences the enzymes cut

» generate restriction fragments: DNA that can be joined to other DNA pieces cut with the same restriction enzyme cut / “digest”

→ enables them to base-pair / “anneal” (DNA ligase solidifies their bond) and form recombinant DNA molecules

eg.

• alul

• BamHi*

• EcoRI*

* has “sticky ends” which refers to how their sequences overhang post-cut

recombinant DNA molecules

DNA molecule formed by splicing together DNA cuttings from one specific restriction enzyme

→ can have DNA from humans and bacteria

DNA molecules that contain DNA from more than one organism

DNA cloning

Making more copies of the DNA

Procedure used to introduce altered/foreign DNA into a recipient cell that will be replicated and passed unto the cell progeny

process involves:

• isolation of insert (DNA fragment) from chromosome, cut by restriction enzymes

• cutting a vector: plasmid that will carry desired DNA fragment into the recipient cell

  • must be cut with the same restriction enzyme

• joining the insert with the vector → recombinant DNA molecule

• introducing the plasmid to a host cell where DNA will replicate

vector

Plasmid or bacteriophage.

Has many features that make it invaluable for cloning, such as:

• origin of replication » enables plasmid to make more copies of itself independent of the chromosome (can be in thousands)

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• multiple-cloning sites » sites that contains multiple restriction sites: recognition sequences of a restriction enzyme

  • gets cut and desired DNA for cloning (inserts) gets inserted here

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• selectable marker » gene that enables researchers to eliminate cells that have not taken up molecules containing the [__] sequences

  • provides the cell immunity to be grow on selective media that are typically anti-microbial » kills cells that don’t have it otherwise

    • eg. ampicillin » NA-Ampicillin is the most common culture media

  • selective environment ensures only bacteria with the [__] grow

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• second marker » helps distinguish cells with successful recombinant plasmid (has the blue insert), from what would be just an intact vector (just red)

Vector pUC18

One of the most common recombinant DNA

• selectable marker » ampicillin resistance

  • grown on an NA-Ampicillin culture media

• second genetic marker » lac’Z gene

  • produces Beta-galactosidase, which interacts with the color-less substrate X-gal added to the media

  • → cleaves it to produce a blue compound

  

  • intact vector (no recombinant) » intact lac’Z → blue

  • broken vector (HAS recombinant) » white

transgenic

Uses for GMOs and DNA cloning:

• DNA production » study genes, identify organisms

• Protein production » human insulin, vaccines, chymosin (rennin) for cheese production via coagulation

• Genetically altered plants » pest-resistant, herbicide-resistant, improved nutrient value, edible vaccines

  • another word for GMO’s like these are [__] organisms

reverse transcriptase

Enzyme produced by retroviruses

• reverse transcription: uses RNA as template to make DNA → cDNA » complementary DNA

• useful for:

  • generating eukaryotic libraries that are intron-free copies of eukaryotic genes

    • introns = noncoding regions, removed in transcription of DNA to mRNA. plasmids in bacteria lack this mechanism

      • if plasmids were given the original DNA, its proteins produced would have faulty proteins produced by introns

    • as mRNA only contains exons, it can be used to reverse engineer to an accurate form of DNA that can be replicated in bacteria

  • gene expression studies

electrophoresis

Technique that separates DNA, RNA, and proteins according to their size

Set up:

• DNA is put into wells in gel

• gel subjected to electric current (two polar charged electrodes at ends)

Result:

DNA migrates towards the (+) electrode as it is negatively charged; smaller fragments migrate faster and closer to the buffer solution. Usually have markers to indicate size

DNA are stained for viewing.

DNA and RNA sequencing

Process of determining the order of nucleotides in DNA and RNA

Next-generation sequencing » much quicker and cheaper

as low as $70, takes a few hours

Sequencing projects:

• Human Genome (1990) » wanted to sequence whole genome of humans

• Human microbiome (2007) » wanted to sequence microbes found on human body

• Earth BioGenome (2018) » effort to sequence genome of all known animals, plants, protozoan, and fungal species

applications

[___] of DNA sequencing can:

help identify genetic alterations that may result in disease

• Sickle cell anemia » single base-pair change in gene → point mutation (substitution)

• Cystic fibrosis » 3 base-pair deletion

• Breast cancer » mutation in BRCA1 or BRCA2 gene

Detect and trace infectious diseases by the CDC

• eg. foodborne illnesses » sequence genome of organism and place it into cloud and track if other people encountered the same gene

Assist in studying evolutionary relatedness of organisms

polymerase chain reaction

Allows exponential amplification of DNA → creation of over a billion copies of a given region of DNA in hours. Exponential (multiplies by 2 per cycle) Can do without culturing.

Reaction requirements:

• Target DNA » serves as template

• Primers: short, synthetic DNA sequences that are complementary to and mark starting point for DNA synthesis

  • determines region of target DNA to amplify

• Taq polymerase: heat-stable, DNA polymerase. assembles new DNA strand by adding nucleotides to primers

• Deoxynucleotides: building blocks used by the enzyme to construct new DNA strands

  • dATP, dGTP, dCTP, dTTP

primers

Short, single-stranded DNA fragments that bind to the complementary sequences in target DNA for replication

• defines target sequence to be amplified and determines length of PCR product

• repeatedly cleaves longer DNA into shorter ones, and using the shorter ones as a template

denaturation

The Three Step Amplification Cycle of PCR:

1. [___]: DNA strands split by heat (~95 C)

2. Annealing: primers anneal/bind to target DNA sequence (~50-60 C)

3. Extension: DNA synthesis by Taq polymerase (~68-72 C)

CRISPR-Cas

[___] technology are gene editing tools that can:

» locate, cut out, replace, or add targeted changes to nucleotide sequence of living cell genome

uses

• Cas9: enzyme that makes double-stranded cut of DNA at a specific location so a new sequence can be inserted

• Guide RNA: pre-designed RNA sequence that recognizes desired DNA sequence

  • placed in Cas9 alongside the insert/DNA fragment that will be inserted

  • guides Cas9 to target sequence

gene therapy

Use of genetic engineering technologies to manipulate DNA in humans to treat genetic diseases

• clinical trials are assessing usage of Cas9 in [__]

• eg. hemophilia has been cured using Cas9