Honors Biology 12.1-12.21: DNA Technology and Genomics

Cas Protein

Prokaryotes have these specialized proteins that function to remove DNA inserted via a viral infection. These work similarly to restriction enzymes in that they cut DNA.

Noncoding DNA

- 19% of DNA consists of introns. - 5% is gene control sequences (promoters), enhancers, and microRNAs (small single stranded RNA that aids in gene expression). - 15% unique, non-repetitive sequences. - 15% repetitive DNA sequences. - Some are short, and some are 1000s of nucleotides long. - Prominent at centromeres and telomeres (ends) of chromosomes. - 44% repetitive DNA that includes transposable elements and related sequences. - Repetitive nucleotide sequences 100s of bases long scatter throughout the genome known as "jumping genes". - Can move to different areas within a chromosome or even move to other chromosomes.

Complementary DNA (cDNA)

A DNA molecule made in vitro using mRNA as a template and the enzyme reverse transcriptase. This molecule therefore corresponds to a gene but lacks the introns present in the DNA of the genome. This is very useful in studying genes with specialized functions in cells such as liver or brain cells.

Recombinant DNA

A DNA molecule that has been manipulated in the laboratory (in a test tube) to carry nucleotide sequences derived from two sources, often different species. This is used for genetic engineering.

Restriction Enzymes

A bacterial enzyme that cuts up foreign DNA (at specific DNA sequences called restriction sites), thus protecting bacteria against intruding DNA from phages and other organisms. These are used in DNA technology to cut DNA molecules in reproducible ways. They only work on the specific associated restriction site.

Agarose Gel

A carbohydrate extracted from seaweed that has a network of carbon-like thread that can act as a molecular sieve (filter).

CRISPR-Cas9

A form/powerful new tool for gene editing. This technique can be used to remove non functioning genes and insert functional version. This system cuts the faulty gene out but also carries a functional version of the gene so DNA repair enzymes insert the functional version.

Vaccine

A harmless variant or derivative, usually a bacterium or virus, of a pathogen used to stimulate a host organism's immune system to mount a long-term adaptive response against the pathogen. These are created on a mass scale using genetically engineered organisms. A gene for a surface protein of a virus is inserted into a vector that will produce large quantities of the protein. Others are genetically modified versions of a live virus (pathogens are removed/altered). Examples of pathogens with these are measles, mumps, and polio.

GMO issue

A lot of these have not been approved to be used for the consumption of humans. There are growing concerns about human use/consumption of this in foods and products. The initial concern was DNA technology could create new pathogens. Strict guidelines were established for the generation of these. The largest concern is the use of these for human consumption. No long term studies have been performed.

Whole-Genome Shotgun

A method for determining the DNA sequence of an entire genome. After a genome is cut into small fragments via restriction enzymes, each fragment is cloned and sequenced and then placed in the proper order. Mapping software assembles the millions of overlapping short sequences in each chromosome. This was developed by J. Craig Venter. It takes roughly 1 hour and costs $1000.

STR Analysis (short tandem repeat)

A method of DNA profiling that compares the lengths of short tandem repeats selected from specific sites within the genome. The database that collects and stores this data is known as the Combined DNA Index System (CODIS). The US legal system requires 13 sites for comparison. The likelihood of 2 separate individuals having the same identical 13 sites is 1 in 10 billion.

DNA Profiling

A procedure that analyzes DNA samples to determine if they came from the same individual. 1) DNA is collected from the crime scene, suspects, victims, or other evidence is isolated. 2) Markers from the DNA sample are amplified (a lot of copies are made after the DNA is cut into fragments using restriction enzymes). 3) The amplified markers are compared showing which samples came from the same source.

Transgenic Organism

A recombinant organism that contains genes from another species.

Short Tandem Repeat (STR)

A series of short DNA sequences that are repeated many times in a row in the genome. Each person has these repeated differently which is what scientists compare that will most likely be different from person to person. Ex: AGAT or GATA

Plasmid

A small ring of independently replicating DNA separate from the main chromosome(s). These are found in prokaryotes and yeasts.

Cas9 Protein

A specific protein that cuts double stranded DNA. This will cut any segment of DNA based on the guide RNA within the protein. Researchers use this to cut specific regions at the beginning and ending of a gene to literally cut the gene out of the genome - known as gene "knock out".

Restriction Site

A specific sequence on a DNA strand that is recognized as a "cut site" by a restriction enzyme.

Gel Electrophoresis

A technique for separating and purifying macromolecules, either DNA or proteins. A mixture of the macromolecules is placed on this between a positively charged electrode and a negatively charged one. Negative charges on the molecules are attracted to the positive electrode, and the molecules migrate toward that electrode because of their negative charge (PO4-). The molecules separate in this according to their rates of migration, which is mostly determined by their size: smaller molecules generally move faster through this because they are able to move through the carbon-threads easier, while larger molecules generally move more slowly because they are caught by the network of carbon-threads. It can also sort based on other physical properties, besides size and electrical charge. When the current is turned on each well, where DNA was inserted has a "lane" of bands which are highlighted by either: photographic film (if the DNA is radioactively marked) and fluorescence (if the DNA is dyed). The thickness of the bands indicated the amount of DNA present. This is an essential component in creating DNA profiles.

Polymerase Chain Reaction (PCR)

A technique used to obtain many copies of a DNA molecule or a specific part of a DNA molecule. In the procedure, the starting DNA is mixed with a heat-resistant DNA polymerase, DNA nucleotides, and a few other ingredients. Specific nucleotide primers flanking the region to be copied ensure that it, and no other regions of the DNA, is replicated during the this procedure. Major component that allows this to happen was the discovery of a heat-resistant DNA polymerase isolated from a bacterium that inhabited a hot springs (otherwise the protein would denature in the hot environment). This process can be used to copy fragments of DNA. It is used to copy DNA up to 3000 years old (mummy) or 40000 year old wooly mammoth DNA. 1) Reaction mixture is heated to separate the DNA double helix. 2) Mixture is cooled enabling the primers to bond to the DNA templates. 3) Heat-stable DNA polymerase extends the primers building new copies of the target DNA sequence.

Gene Therapy

A treatment for a disease in which the patient's defective gene is supplemented or altered. Currently the success rate is fairly low. Researchers don't see any ethical issues transplanting genes as a form of the this - it's viewed as an equivalent to an organ transplant. Ethical issues arise when researchers consider manipulating genes in gamete forming cells or a developing zygote. The train of thought is that genetic defects could be eliminated in children and their descendants. This can affect future generations and is seen as a potential gateway to eugenics - the deliberate effect to control the genetic make up of human populations. Used to treat various disorders: - Leber's congenital amaurosis (leads to blindness) - Severe combined immunodeficiency (bubble boy immune system disorder) - Nervous system and blood disorders

Uses of GMOs

Add nutritional value to crops. - Boost beta-carotene levels in rice (used to make vitamin A). - Milk proteins for rehydration formulas added to rice. Increase nutritional value in livestock. - Decrease levels of unhealthy fatty acid and increase omega-3 fatty acids in pork. Boost production time and quality of animal products. - Increase quality of wool in sheep. - Increase growth rate of cows and salmon.

Reverse Transcriptase

An enzyme encoded and used by retroviruses that catalyzes the synthesis of DNA on an RNA template. The presence of introns creates an obstacle for researchers looking to isolate eukaryotic genes. Prokaryotes are used to clone these genes however prokaryotes do not have the machinery to remove introns. To avoid this, the mRNA transcript of the eukaryotic gene is isolated. 1) The mRNA transcript of the desired gene is isolated from the cytoplasm of the eukaryote. 2) This enzyme is used to create a single stranded DNA transcript of the gene. 3) The mRNA transcript is broken down using another enzyme. 4) DNA Polymerase is used to synthesize the complimentary strand to the single stranded DNA transcript.

DNA Ligase

An enzyme, essential for DNA replication, that catalyzes the covalent bonding of adjacent DNA polynucleotide strands. This is used in genetic engineering to paste a specific piece of DNA containing a gene of interest into a bacterial plasmid or other vector.

Human Genome Project (HGP)

An international collaborative effort to map and sequence the DNA of the entire human genome. This began in 1990 and was completed in 2003. It sequenced 99% of the human genome with 99.999% accuracy. The missing 1% is the "gap" in the human genome that is not clearly understood. As in most eukaryotes, on about 1.5% of human DNA is contained in genes that code for proteins, tRNAs, or rRNAs. Most eukaryotic DNA (98.5% in the case of humans) is noncoding DNA. Researchers believe that noncoding regions of DNA do have significance, but they are unsure of what it is yet. Ex: Humans, rats, and mice have 500 noncoding regions of DNA that are identical in sequence among the 3 species.

Genetically Modified Organism (GMO)

An organism that has acquired one or more genes by artificial means. If the gene is from another species, the organism is also known as a transgenic organism. This includes lots of crops. Ti plasmid from Agrobacterium tumefaciens is a commonly used plasmid for creating these. 1) Restriction enzymes and DNA ligase are used to insert the desired gene into the plasmid. 2) The recombinant plasmid is put into a plant cell and the DNA integrates into the plant cell's chromosomes. 3) Recombinant cells are cultured and grown into a plant.

Clone

As a verb, to produce genetically identical copies of a cell, organism, or DNA molecule. As a noun, the collection of cells, organisms, or molecules resulting from this; informally, a single organism that is genetically identical to another because it arose from the cloning of a somatic cell.

Diagnosing Diseases

DNA technology is used to identify diseases (sickle cell, cystic fibrosis, hemophilia, Huntington's). It is frequently done before symptoms have onset or even before birth. It is also used to identify specific strains of bacteria or viruses enabling scientists to determine the source/origin of the bacteria or virus. - Source of outbreaks can be determined by DNA analysis. Used for: - E. coli - Listeria - HIV

Inserting Genes

Genes for desired traits in certain organisms can be this into the genome of organisms of a different species that could benefit from having that gene. This can be done to essentially turn them into protein factories generating a desired protein that is coded for by the inserted gene. Ex: Round up ready (resistant) crops Ex: Human insulin for diabetics

Nucleic Acid Probe

In DNA technology, a radioactively or fluorescently labeled single-stranded nucleic acid molecule used to find a specific gene or other nucleotide sequence within a mass of DNA. The probe hydrogen-bonds to the complementary sequence in the targeted DNA. This is used when the desired nucleotide sequence for a gene is already known.

Vector

In molecular biology, a piece of DNA, usually a plasmid of a viral genome, that is used to move genes from one cell to another.

GMO Labels

In the US, odds are you have eaten some genetically modified produce and it does not have this in any fashion. - Corn, soybeans, or their by-products (high fructose corn syrup). Most countries require GMO foods to have these so consumers have the choice.

Benefits of HGP

Mapping the human genome enables scientists to identify disease-associated genes (some cases of Parkinson's and Alzheimer's). Scientists can also compare genes found in multiple species which might better help them understand how the gene functions. Ex: The same gene exists in humans, rats, and zebra finches - Humans = altered versions can lead to Parkinson's or Alzheimer's, rat = sense of smell, and zebra finches = song recognition.

DNA Technology

Methods used to study and/or manipulate DNA, including recombinant DNA technology.

Restriction Fragments

Molecules of DNA produced from a longer DNA molecule cut up by a restriction enzyme. These are used in genome mapping and other applications. They have "sticky ends."

Repetitive DNA

Nucleotide sequences that are present in many copies in the DNA of a genome. The repeated sequences may be long or short and may be located next to each other (randomly) or dispersed in the DNA. They lie in between genes in humans. It varies person to person drastically.

GMO Study

Pigs: - In 2012 a study on pigs and these crop consumption was performed. - Short and long term measurements were taken. - No significant difference between 2 research groups and no foreign DNA was found in the pigs. Children: - In 2012, a study on Chinese school children was performed. - 3 study groups each having a different source of beta-carotene: 1) Spinach (natural) 2) Golden rice 3) Capsule of pure beta-carotene - The children consuming the golden rice containing these have comparable levels of Vitamin A as the children receiving the capsule of pure beta-carotene. - No health or wellness issue came from this study but this raised huge ethical issues among both Chinese and American authorities. - The study was limited to only observing the possible short term effects. - It's still not possible to determine the long term effects (if any) on the consumption of these foods.

Glycoprotein

Require mammalian cells to be produced as they are required to be attached to the appropriate chain of sugars to the protein. - Tissue plasminogen factor (TPA) - heart attacks - Human erythropoietin (EPO) - RBC production To produce these required human proteins/products, these genes are inserted into different mammals (goats and sheep).

Third Generation Sequencing

Researchers are working so very large/long segments of single stranded DNA can pass through a nanopore and an electrical current can detect which nitrogenous base is present as it passes through the nanopore. The device is the size of a candy bar and it hooks up to a USB port. It enables companies like 23 & me and ancestry.com.

Primers

Short, artificially created, single-stranded DNA molecules that bind complimentary to each end of a target sequence during a PCR procedure. They are 15-20 nucleotides long. Two are used for the PCR procedure.

Next-Generation Sequencing

Small segments of DNA (roughly 300 nucleotides long) can be sequenced using this technology (developed in 2001). 1) A single stranded DNA sequence is isolated. 2) DNA polymerase and other reagents are added to the sample to synthesize the complementary strand. 3) This machinery is able to detect which nucleotides are used to produce the complementary strand enabling determination of the sequence. This is a very inexpensive method to sequence billions of nucleotides in a day.

Genetic Engineering

The direct manipulation of genes for practical purposes. Bacteria that has been this is used to produce a wide array of chemicals, such as cancer drugs, pesticides, and medicines. It is also used to transfer genes from one animal species to another.

Biotechnology

The manipulation of living organisms or their components to make useful products. It is an ancient practice and has been used for yeast making beer and selective breeding for livestock or other animals.

DNA Cloning

The production of many identical copies of a specific segment of DNA. It is used for the production of useful products through genetic engineering. The biggest issue is finding the segment of DNA that codes of the desired gene. To start this process, it requires a bacterial plasmid to act as the vector and the segment of DNA that is the desired gene. This creates genes that can be inserted in other organisms and harvests proteins that are created by the organisms with the inserted gene.

Gene Cloning

The production of multiple copies of a gene.

Environmental Safety

These are concerns that transgenic crops may pass their new genes on to related wild species which can disturb the natural ecosystem. - GMOs introduced to ecosystems can become invasive. - Plants considered weeds could evolve to become more resistant to herbicides thereby making practically impossible to naturally grow crops. Research into unintended gene transfer has shown to cause no detrimental effects to the environment. Advocates have shown that some GMO existence in an environment can actively help. - Bacteria engineered to breakdown oil spills.

Forensics

The scientific analysis of evidence for crime scene and other legal proceedings. DNA profiling is used in this field in a number of ways: 1) Evidence proving a suspect of guilt. 2) Evidence exonerating a person wrongfully convicted. 3) Identifying injured/diseased victims. 4) Determining biological paternity. 5) Analysis of nonhuman materials - poaching prevention.

Genomics

The study of complete sets of genes (genomes) and their interactions. Advances in DNA technology has enabled the expansion of this field - started with sequencing the entire genome of meningitis and pneumonia - 1,709 genes. Baker's yeast was the first type of eukaryotic cell to have its full genome sequenced - 6275 genes. In general, prokaryotes have smaller genomes than eukaryotes. The size of a eukaryotic genome does not correspond to the complexity of the organism.

Proteomics

The study of whole sets of proteins encoded by genomes and their interactions. This allows scientists to determine what cells produce which proteins and when they're produced to understand their functionality in an organism.

Bioinformatics

The use of computers, software, and mathematical models to process, store, apply, and integrate biological information from large sets of data. GenBank is a massive compilation of DNA information with a number of tools that allow for easy and free nucleotide sequence comparisons. - Researchers can enter a nucleotide sequence from an organism and find similarities in other species. - This can help determine genetic relatedness in the evolutionary tree.

Treating Diseases

There are 2 widely used products made using recombinant DNA. 1) Human insulin - diabetics 2) Human growth hormone (HGH) - dwarfism or other hormonal issues. Inserting these genes into bacterial vectors allows the rapid production and harvesting of the products. Plants can also be used as vectors. - Environmental conditions are not as stringent as animal or bacterial cell vectors. - Carrot cells are used as vectors to produce a fat hydrolyzing enzyme to treat Gaucher's disease (rare genetic condition).

Gene Editing

This has been successfully done in mice. - 2014 tyrosinemia (effects tyrosine metabolism) caused gene was replaced with a functional gene - 2015 dystrophin (effects muscle development) causes gene replaced with a functional gene. Human trials have taken place in China only very recently. The most recent/controversial trial was on human babies for HIV resistance.

Isolating DNA

This requires manipulating the DNA. Restriction enzymes are used for cutting specific sequences of DNA known as restriction sites. The DNA is cut in a staggered fashion, not straight through, and the fragments produced are known as restriction fragments with "sticky ends." Restriction enzymes only work on the specific associated restriction site. There are 1000s of different restriction enzymes.

Mass Producing Genes

Using recombinant cells to mass produce a desired protein is largely done via prokaryotic bacteria (usually E. coli) for a number of reasons: - Plasmids available for gene cloning vectors - Rapid growth & reproduction rate - Coss effective mass storage - Engineered to mass produce the specific protein and even secrete it into the growth medium for easy collection Used to produce a number of human drugs and enzymes necessary for food production. Often eukaryotic cells are required to produce a protein. Yeast is the "go to" eukaryotic choice for the production of a number of proteins: - Antimalarial drugs - Hepatitis B vaccine - Interferons - treatments for cancers and viral infections