Bio Test 2

Genetic Engineering in : Agriculture (Transgenic Plants)

- Increase yields of crops by engineering plants with increased nutrients, plant resistance to pests, disease, herbicides, and environmental stress - Use plants as bioreactors for human pharmaceuticals - Decrease crop loss due to pests reduced by introducing engineered strains of insects that do not afflict certain plants

In nature, approximately ____ of bacterial are able to pick up DNA from the surrounding environment DNA acquired is typically a small fragment that gets incorporated into the genome

1%

Gene Manipulation

1)DNA Extraction ---> DNA must be removed from cells with little damage and purified for storage and use of DNA 2) Gene Isolation ----> Gene of interest must be isolated from the rest of the genome ---- Restriction Enzyme Digest ----> use a restriction enzyme that cuts AROUND the gene and isolate the DNA fragment using gel electrophoresis ---Polymerase Chain Reaction ----> use gene specific primers to make multiple copies of the gene of interest 3) Gene Modification ----> Changes can be made to the gene itself in addition to being paired with a promoter, terminator and selectable marker (take populations of cells and tells you which ones you have change, and which ones you haven't) (ex: "can you survive in a presence of an antibiotic" if you can then you have been genetically modified) This process is most commonly described in regards to plasmid engineering, but the basic steps are the same for manipulating a gene construct to be inserted into any organism

What are the 3 major differences between Genetic Engineering and Gene Therapy?

1)The term "gene therapy" is usually reserved for the process of genetically modifying humans; while modified plants/animals/bacteria/fungi are "genetically engineered." Note: DNA molecules are genetically engineered in the laboratory. 2)The process of genetically engineering plants and animals usually takes at the earliest stages of life (embryonic stage); while gene therapy is a process of genetically modifying an adult orc child (but long after embryonic stage). The process of genetically altering human sperm, egg or embryo is usually referred to as Germ-line Gene Therapy. 3)A genetically engineered organism is entirely genetically modified - all cells contain the altered DNA - even if a new trait (or protein) is expressed in one tissue (like human protein made in goat's milk); while gene therapy changes the DNA of some cells of an adult person (lung cells, skin cells, heart cells, etc.) making them a chimera.

EcoR1 cuts ONLY when the enzyme encounters this DNA sequence: (Don't forget...DNA is composed of 2 antiparallel strands)

5' G A A T T C 3' would become : 3' C T T A A G 5'

Recombinant DNA

A DNA molecule that is made of segments of DNA from more than one source

Restriction Site

A specific sequence on a DNA strand that is recognized as a cut siteby a restriction enzyme. When DNA is cut across both of the two strands, EcoR1 cuts AFTER the G in the sequence GAATTC

Transformation of plants is commonly done using a bacteria called _______________________ to mediate the transfer of the genetic material

Agrobacterium

When looking at a STR analysis. What do we look at on a chart to determine male or female?

Amelogenin

DNA polymerase

An enzyme that catalyzes the formation of the DNA molecule. Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule Principle enzyme in DNA replication

Why is palidrome significant?

Because the DNA is cut across both of the two strands, EcoR1 cuts AFTER the G in the sequence GAATTC ^ THAT IS CALLED RESTRICTION SITE now if we want to, we can attach (or insert) a new foreign piece of DNA that has the SAME ENDS. These single stranded, matching ends are called "STICKY ENDS" this joining requires a different enzyme called "DNA LIGASE"

CAR-T cells

Chimeric Antigen Receptor Modified T-Cells are designed to target a specific receptor found on the B-Cell of patients suffering from leukemia The drug Kymriah, is approved in the united state for the treatment of patients up to 25 years of age with B-cell precursors ALL that is refractory in second or later replase

CODIS

Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1998 by the FBI Links all 50 states, doesn't stay within local system goes across country. Requires data for 20 core STR markers This is an increase in data points from the original 13 effective 2017

How does gel electrophoresis work?

DNA fragments move through a gel (jello-like) material The gel material for DNA analysis is typically agarose The DNA movement through the gel is accomplished by applying an electrical current to the gel Since DNA is NEGATIVELY charged, it is drawn toward the positive electrode in a gel electrophoresis tank The distance a DNA fragment travels through the gel is dictated by the size of the fragment shorter fragments travels further faster than bigger ones. (ex : motorcycle vs bus) A fluorescent chemical mixed in the gel, SyBr-Green, binds to DNA and "glows" under UV light. This allows us to visualize the DNA in the gel and photograph the data

Why doesn't ecor1 cut up e.coli's own dna?

E.coli protects its own DNA with a chemical modification called METHYLATION. This chemical "signature" prevents the enzyme EcoR1 from cutting the DNA. Bacteriophage DNA is susceptible to be cut up because its DNA is usually unmodified (or unprotected).

Germ-Line Therapy is the stage when gene therapy is done at...

EARLY : Sperm or Egg or Embryo which PERMANENTLY changes genes of an individual AND his/her descendants)

Genetic Engineering in : Medicine (Gene Therapy & Cloning)

Eliminate approximately 2,000 single gene human diseases and reduce genetic predispositions (diagnostics) to certain diseases in humans Human therapeutic (embryonic stem cells) and reproductive cloning

Bioremediation

Environmental Bioremediation Genetically engineered organisms used to clean up hazardous waste, landfills and environmental disasters (i.e. Exxon Valdez oil spill in Alaska, Gulf Oil spill) Use of living organisms such as prokaryotes, fungi, or plants to detoxify polluted ecosystems.

Do identical twins have the same DNA?

Everyone has their own unique DNA, except Identical Twins. They start off with the same exact DNA... but as mutations occur naturally as DNA replicates so as they get older it changes.

How many peaks would a female have looking at a STR analysis?

Females are XX Females have one peak and the next would be black

The ability to do this led to a revolution in DNA science and technology called ?

Genetic Engineering

The permanent change to the genetic makeup or an organism is ?

Genetic Engineering

Genetic Engineering in : Environmental Bioremediation

Genetically engineered organisms used to clean up hazardous waste, landfills and environmental disasters (i.e. Exxon Valdez oil spill in Alaska, Gulf Oil spill)

What is Geneotype

Genotype is the genetic composition of an organism Genotype = Genes Both start with G

Gene Therapy at the sperm, egg, or early embryo stage is called

Germ-line Therapy which PERMANENTLY changes genes of an individual AND his/her descendants)

Males are (XY) which means

Heterozygous for amelogenin

Benefits of Genetic Engineering on : Transgenic Animals

Improved efficiency and quality of production - meat, milk, wool, nutrient - pregnancy/reproduction rate Increase resistance to diseases Extended storage properties (increased shelf-life) Ability to produce human pharmaceuticals Animals are used as models for human diseases (ex : lab rats, & animal testing) Organ donors for xenotransplantation

CRISPER-Cas9

In 2011 : Clustered regularly interspaced palindromic repeats (CRISPR) were discovered in bacteria and archaea. - They are used to increase immunity to invading viruses by cleaving viral DNA and inserting pieces of that DNA into their own genome. They then transcribe this DNA into RNA and combined it with Cas9 proteins to make double strand breaks in the invading viral DNA. - In a lab setting, scientists pair a specific guide RNA (gRNA) with the Cas9 Protein which allow the complex to target a specific region of the DNA - Cas9 protein then causes a double strand break in the DNA, allowing for various techniques to be used to edit the DNA at that location.

How many peaks would a male have looking at a STR analysis?

Males are XY. Males have 2 peaks.

Genetic Engineering

Process of making changes in the DNA code of living organisms EcoR1 recognizes the base sequence, but cuts the backbone of DNA. Two foreign pieces of DNA can be joined together after Restriction Enzyme Digest. Product is a recombinant (or genetically engineered) DNA molecule. Another is example is how we make insulin to help those with diabetes, so using a restriction enzyme we isolate the gene for insulin and then we put that into a bacteria to make human insulin

What is NOT a motivation for the production of transgenic animals?

Reduce the nutrient quality of products

What is one way to distinguish individuals in a Forensics case (DNA Profiling).

Restriction Fragment Length Polymorphism (RFLP) analysis can also be used to detect or identify small changes in someone's DNA (compared to other people)

Bacteriophage

Restriction enzymes RESTRICT the replication of bacteriophage (bacterial virus)

Why do bacteria contain restriction enzymes?

Restriction enzymes RESTRICT the replication of bacteriophage (bacterial virus) Restriction digest is the phrase used to describe the breakdown of DNA molecule by a restriction enzyme. Since their initial discovery in the 1970's, over 100 restriction enzymes have been discovered. Restriction enzymes are named according to their bacterial source EcoR1 ----> E. coli

sticky ends

Single stranded ends of DNA left after cutting with enzymes This is when we can attach (or insert) a new foreign piece of DNA that has the SAME ENDS.

chimera

TWO DIFFERENT A chimera is a single organism composed of genetically distinct cells. This can result in male and female organs, two blood types, or subtle variations in form. Animal chimeras are produced by the merger of multiple fertilized eggs. Normally, chimerism is not visible on casual inspection. in genetics, an organism or tissue that contains at least two different sets of DNA, most often originating from the fusion of as many different zygotes (fertilized eggs). Another way that chimerism can occur in animals is by organ transplantation, giving one individual tissues that developed from two genomes. For example, a bone marrow transplant can change someone's blood type. In 1980 - a sheepgoat was made chimera was made (geep) A genetically engineered organism is entirely genetically modified - all cells contain the altered DNA - even if a new trait (or protein) is expressed in one tissue (like human protein made in goat's milk); while gene therapy changes the DNA of some cells of an adult person (lung cells, skin cells, heart cells, etc.) making them a chimera.

What is genetic engineering?

The direct manipulation of an organism's genes by technological intervention/manipulation It is a phrase used to describe some modern techniques in molecular biology, for example, recombinant DNA technology Technology used to alter the genetic materials of living cells and organisms in order to make them capable of PRODUCING NEW SUBSTANCES or PERFORM NEW FUNCTIONS We can change the DNA which can result in changing the protein it produces This is a PERMANENT CHANGE to the genetic makeup of an organism

What is the primary method of gene therapy?

Transduction

Transduction

Transduction (aka viral transfection) is the process of inserting foreign DNA into a cell via a virus or viral vector This is one of the primary mechanisms used in gene therapy Viral vectors are modified by removing some of its virulence (infection) capabilities and inserting the new gene to be delivered to the patient's cells Transduction also occurs in nature HIV and Adenovirus are two types of viruses that integrate their genetic material into the host genome

Transfection

Transfection is the general term for the insertion of foreign DNA into animal cells - there are multiple techniques that can be used to accomplish this. Microinjection is the process of injecting DNA directly into the nucleus - typically done with an egg or embryo in vitro Lipofection is the process of using a liposome as a vector to deliver DNA into cells in culture Electroporation is the process of delivering a brief electric shock to cells in culture, which creates small holes in the cell membrane, allowing for the uptake of DNA particles

The process of an organism taking up DNA from the environment.

Transformation

Biolistics (Gene Gun)

Transformation of Plants : Gold or tungsten particles are coated with DNA and shot into young plant cells or plant embryos in culture When the particles pass through the cells, the DNA is left behind. Some of that genetic material will enter the cell and be incorporated into the genome This method can be used on plants that are not susceptible to Agrobacterium infection Can damage cells

Agrobacterium

Transformation of plants is commonly done using a bacteria called Agrobacterium to mediate the transfer of the genetic material The gene of interest is inserted into a Ti plasmid, which is inserted into the Agrobacterium Plant tissue cells are then cultured with the Agrobacterium. Using conjugation, the bacteria transfers a segment of DNA from the plasmid containing the gene of interest (T-DNA) into the plant cell The T-DNA is piloted to the nucleus and is integrated (semi-randomly) into the plant cell genome

An adult who receives gene therapy to treat a disease will still have the possibility of passing the disease onto their offspring ?

True

One example of Gene Therapy?

Using HIV to Kill Cancer

Amelogein

What we look for when looking at an STR analysis if female(homo) 1 peak with a black one next to it(XX) if male (hete) 2 peaks (XY)

Competent

When an organism has the ability to be transformed, take up DNA from the environment, then the organism is then considered competent

are STR (Short Tandem Repeats) NONCODING?

YES STR IS NON CODING

CRISPR

a collection of DNA sequences that tells Cas9 exactly where to cut CRISPR is a family of DNA sequences found within the genomes of prokaryotic organisms such as bacteria and archaea.

Why do we use Gel Electrophoresis

a method for analyzing DNA

Arabinose

a monosaccharide that activates the promoter in the Arabinose operon

Plasmid

a small, circular piece of DNA which contains a few genes . Separate form the chromosomes.

One method of inserting DNA into cells is the use of a virus as

a vector, or transporter, of the genetic material

Heterozygous

alleles differ and can be resolved

Homozygote

both alleles are the same length

Bacterial transformation is the process of ?

changing the genetic make-up of an organism by passing genetic material directly through the cell membrane

When does Ecor1 cut after ?

cuts after the G in the sequence : GAATTC

Where can DNA differences be found?

differences can be found if we examine the number and length of the DNA fragments produced from a restriction enzyme digestion.

DNA ligase

enzyme that chemically links DNA fragments together

Agarose

gel material for DNA analysis

2 peak per loci

heterozygous Males are heterozygous for amelogenin (XY) Males have 2 peaks.

1 peak per loci

homozygous Females are homozygous for amelogenin (XX) Females have one peak and the next would be black

palidrome

how the DNA strand that EcoR1 cuts for ONLY examples : racecar, mom,2002

electroporation

is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell CELL ---> INCUBATION ---> HEAT SHOCK----> RECOVERY <3

DNA Liagase

is an enzyme which can connect two strands ofDNA together

Restriction Enzymes (and DNA Ligase) can be used to?

manipulate DNA - to cut and paste (or join) foreign pieces of DNA together. (genetic engineering)

Polymorphism in people's DNA

many shapes or kinds normally occurring differences in our DNA sequence

What is Phenotype

phenotype is the physical appearance of an organism Phenotype = Physical Both start with P

Cas9

protein that cuts DNA

DNA ligase

put fragments back together

The joining of sticky ends .....

requires a different enzyme called "DNA LIGASE"

Ligase is knowing for

sealing the gap in when the sticky ends meet

Restriction enzymes cut _______________ dna sequences

specific

Bio-mimicry

the design and production of materials, structures, and systems that are modeled on biological entities and processes.

Gene Therapy

the process of genetically modifying humans; while modified plants/animals/bacteria/fungi are "genetically engineered." Note: DNA molecules are genetically engineered in the laboratory.

STR - Short Tandem Repeats

the repeat region is variable between samples while the flanking regions are constant They are NONCODING DNA. 2 - 10 base pairs long Has short sequences of bases that repeats a number of times. Homozygote = both alleles are the same length Heterozygous = alleles differ and can be resolved from one another (has nothing to do with dominant or recessive ^ ) There is original 13 CODIS core STR loci with Chromosomal Positions but there is 20.

Using a restriction Enzyme to make insulin

using a restriction enzyme we isolate the gene for insulin and then we put that into a bacteria to make human insulin to help those with diabetes.