CH. 9 micro

Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products.

** Antibiotic-markers on plasmid vectors are used to identify cells containing the engineered vectors by direct selection. **DNA- short segments of single stranded DNA that are complementary to the desired gene. If it finds a match, it will adhere to the target gene. radioactively labeled **Gene- usually inducible promoters are used to allow expression of gene independent of host cell gene expression

Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products.

** Antibiotic-markers on plasmid vectors are used to identify cells containing the engineered vectors by direct selection. **DNA- short segments of single stranded DNA that are complementary to the desired gene. If it finds a match, it will adhere to the target gene. radioactively labeled **Gene- usually inducible promoters are used to allow expression of gene independent of host cell gene expression

cDNA

complementary DNA are copies can be made from tRNA, mRNA, rRNA also other forms of RNA

reverse transcriptase is best known for?

the replication of HIV

site-directed mutagenesis

a targeted and specific change in a gene

What is a primary participant in cloning an isolated gene>

restriction endonuclease, vector, host organism

TI plasmid

occurs in Agrobacterium tumefaciens; Can be used to introduce rDNA into a plant

What is a RFLP

restriction fragment length polymorphisms- they are the differences in the cutting pattern of specific restriction endonucleases

What are the fragments of the restriction endonucleases called?

restriction fragments

Recombinant DNA Technology (rDNA)

refers to DNA that has been artificially manipulated to combine genes from two different sources. One recombinant DNA technology of great use today is the amplification of DNA (making thousands or more copies of the same DNA molecule).

The function of ligase is to?

rejoin segments of DNA

What is the purpose of a genomic library?

To maintain and retrieve DNA clones

DNA probe is

a piece of DNA; a known sequence

gene probes are

specially formulated tracers

Compare and contrast biotechnology and recombinant DNA technology.

**Biotechnology- the purposeful application of biology principles to industrial and agricultural production - commercial production of: foods (e.g. cheese, yogurt), antibiotics, vitamins and fermentation products **Recombinant DNA Technology (rDNA) Precise manipulation of genes by artificial techniques - almost no limit to gene construction

Explain how rDNA technology can be used to treat disease and to prevent disease.

-Human hormone production -Subunit vaccines; contain only the protein portion of pathogen -DNA vaccines; plasmids encode a viral protein under transcriptional control of promoter region -Gene Therepy; replace defective/mutated genes

Desirable features in a microbial cloning host>

1. rapid turnover fast growth rate 2. can be grown in large quantities using reg culture methods 3. non pathogenic 4. genome that is well delineated

What criteria must a vector meet?

-Self replication -Manipulatable size (small) -Preservation

What is Southern blotting?

A hybridization technique where a sequence of a mutated gene is used as a diagnostic tool.

T/F A nucleic acid probe can be used to id unknown bacteria or viruses in clinical samples?

TRUE

Diagram DNA fingerprinting, and provide an example of its use.

1 collect DNA sample (blood, hair, etc.) 2 fragment DNA with restriction enzymes 3 "load" fragments into a gel, run an electric current -DNA is (-) and will move to the (+) end 4 smaller fragments move faster creating a band pattern on the gel, then you read the pattern

Describe 5 ways of getting DNA into a cell

1. Transformation 2.Electroporation 3.Protoplasts 4.Protoplast fusion 5.Microinjection

Outline genetic engineering with Agrobacterium.

1. the plasmid is removed from the bacterium, and the T-DNA is cut by a restriction enzyme 2. foreign DNA is cut by the same enzyme 3. the foreign DNA is inserted into the T-DNA of the plasmid 4. the plasmid is reinserted into a bacterium 5. the bacterium is used to insert the T-DNA carrying the foreign gene into the chromosome of a plant cell 6. the plant cells are grown in culture 7. a plant is generated from a cell clone. all of its cells carry the foreign gene and may express it as a new trait

Outline the steps in PCR, and provide an example of its use.

1.denaturation: DNA incubated at high temp (94 C) which breaks hydrogen bonds of DNA 2. priming: DNA incubated at relatively low temp allowing primers to attach to single stranded target DNA 3. extension: DNA incubated at intermediate temp, DNA polymerase rapidly replicates DNA 4. DNA present doubles after each round. After 30 rounds, amplified by more than a billion times.

Identify the roles of a clone and a vector in making recombinant DNA

A desired gene is inserted into a DNA vector such as a plasmid or viral genome. The vector will insert the DNA into a new cell, which is grown to form a clone. The idea is that large quantities of the gene or the gene product can be harvested from the clone.

What is gene silencing?

A natural mechanism to inhibit gene expression. Small interfering RNA target a particular gene, and then bind to mRNA causing its enzymatic destruction, silencing its expression.

What is the value of restriction enzymes in recombinant DNA technology?

A special class of DNA-cutting enzymes, used to produce DNA fragments with sticky ends

What sequence when combined w/ its complement, could be clipped by an endonuclease?

AAGCTTTTCGAA

Why do RFPLs result in a DNA fingerprint?

Because it is only a fragment of the DNA

Differentiate biotechnology and recombinant DNA technology.

Biotechnology- the use of microorganisms to make a product rDNA- genetically engineering microorganisms to produce chemicals

T/F In order to dectect recombinant cells, plasmids contain antibiotic resistance genes?

TRUE

Restriction enzymes

Create blunt ends or sticky ends (staggered cuts)

Single nucleotide polymorphisms are found in?

DNA

List one advantage of modifying each of the following: E.Coli, Saccharomyces cerevisiae, mammalian cells, plant cells

E.Coli-easily grown, researchers are familiar with it Saccharomyces cerevisiae-best understood eukaryotic genome/Workhorse of biotechnology. mammalian cells-make protein products for medical use. plant cells-painkillers, synthetic rubber, sunscreen, vaccines, antibodies, large scale-low cost.

PCR step 3

Extension- increase temp to 72C add DNA polymerase and nucleotides. two complete strands of DNA are produced

T/F A DNA fragment w/ 450bp will be closer to the top (neg pole) of an electrophoresis gel than one with 2500 bp?

False- 450 bp will migrate further toward positive pole

T/F The synthetic unit of the polymerase chain reaction is the replica?

False- its the amplicon

T/F Plasmids are the only vectors currently available for use in recombinant procedures?

False- plasmids and bacteriophages are commonly used as cloning vectors.

Identify two advantages and two problems associated with genetically modified organisms.

Good: Less pesticide use, lower costs and higher crop yield Bad: moral issues, super-weeds, cross-pollination

Why isn't cDNA (complementary DNA) synthetic?

It is produced from an mRNA template with the use of reverse transcriptase enzymes from an artificial gene containing only exons.

Of what value is the plant pathogen Agrobacterium?

It naturally produces a vector plasmid for plants: Ti plasmid. . Can be used to introduce rDNA into a plant.

Clone

Population of cells arising from one cell, each carries the new genes

For what is each of the following used in PCR: primer, DNA polymerase, 94 degrees C?

Primer- to attach DNA polymerase- to replicate 94- to break the H bonds

PCR step 2

Priming- add primers that bind to the complementary strand of DNA

Differentiate an RFLP from a gene.

RFLP are variations in the length of DNA fragments produced when a specific restriction endonuclease acts on different DNA sequences. A gene is a specific site on a chromosome that provides info for a certain cell function

Enzymes called _____ _____ can clip a strand of DNA crosswise in selected positions of a polynucleotide

Resctriction endonucleases

Define restriction enzymes, and outline how they are used to make recombinant DNA.

Restriction enzymes cut specific sequences of DNA; destroy bacteriophage DNA in bacterial cells; cannot digest (host) DNA with methylated cytosines. if phages were used to infect bacteria other than their usual hosts, restriction enzymes in the new host deserted almost all phage DNA. restriction enzymes protect a bacterial cell by hydrolyzing phage DNA. the bacterial DNA is protected from digestion because the cell methylates some of the cytosines in its DNA.

Compare selection and mutation.

SELECTION: culture a naturally occurring microbe that produces the desired product MUTATION: mutagens cause mutations that might result in a microbe with a desirable trait; site-directed mutagenesis: change a specific DNA code to change a protein

How are selection and mutation used in biotechnology?

Selection- artificial to select desirable breeds of animals or strains of plants to cultivate Mutations- able to increase fungi penicillin production by 1000x.

Vector

Self replicating DNA used to carry the desired gene to a new cell

How are shotgun sequencing, bioinformatics, and proteomics related to the Human Genome Project?

Shotgun- technique for determining the nucleotide sequence in an organism by sequencing small pieces of the genome Bioinformatics- science of understanding the function of genes with computer analysis Proteomics- science of determining all of the proteins in a cell

List the advantages of, and problems associated with, the use of genetic engineering techniques

Strict safety standards are used to avoid accidental release of genetically engineered microorganisms. -Genetically-engineered crops must be safe for consumption and for release in the environment.

Discuss the value of the Human Genome Project

The human genome project is another beginning process of genetic engineering. It will provide tools for diagnosis and possible repair of genetic diseases

In one sentence, describe how a vector and clone are used.

The vector is the "carrier" to the new cell and the clone "spreads" the new genes

Why do RFLPs (restriction fragment length polymorphisms) result in a DNA fingerprint?

They are specific fragments of digested DNA, which holds a specific code, and are separated by gel electrophoresis.

Why is a vector used in recombinant DNA technology?

They serve as vehicles for the replication of desired DNA sequences.

How are recombinant clones identified?

Through colony hybridization, which uses a DNA probe that is complementary to the identified gene

Contrast the five ways of putting DNA into a cell.

Transformation- cells take up DNA from surroundings Electroporation- electric current forms microscopic pores in the cell membrane Protoplast Fusion- natural recombination from DNA of 2 parent cells through the fusion of protoplasts Inject DNA- using a gene gun through thick cellulose walls Microinjection- for animal cells, micropipet punctures plasma membrane

colony hybridization

Use DNA probes: short segments of single-stranded DNA complementary to the desired gene

Shuttle Vector

Vector that exists in several different species and can move cloned sequences among various organisms

List the properties of vectors.

Vectors serve as vehicles for the replication of desired DNA sequences. 1.most important property of a vector is self-replication, and once in a cell, the vector must be capable of replicating. 2.Vectors must be able to recognize sequence for a restriction enzyme, a unique genetic marker that we can identify once enters the host cell 3.should be small in comparison to host chromosome 4. Plasmids, artificial chromosomes, and viruses are used as vectors.

What is Southern blotting?

a procedure for identifying specific sequences of DNA, in which fragments separated on a gel are transferred directly to a second medium on which detection by hybridization may be carried out.

supression of genes

antisense DNA

Differentiate cDNA from DNA.

cDNA is spliced mRNA that is reverse transcribed into DNA. DNA is not in the reverse order.

Diagram the Southern blotting procedure, and provide an example of its use.

can be used to locate a gene in a cell. Genetic screening uses Southern blotting to look for mutations responsible for inherited diseases in humans.

PCR step 1 CYCLE 1

denaturation- heat target DNA to 94C to separate strands then cool to b/t 50C and 65C= strands stay seperated

Proteomics

determining proteins expressed in a cell

reverse genetics

discovering gene function from a genetic sequence

What is not essential to carry out the polymerase chain reaction?

gel electrophoresis

One way of producing readable patterns of DNA fragments is through?

gel electrophoresis--samples are placed in compartments in soft agar gel and subjected to an electrical current

Examples of current protein products from recombinant DNA technology

immune tx, hormones, enzymes, vaccines

RNAi

inserts DNA encoding siRNA into a plasmid and transferred into a cell

complementary DNA (cDNA)

is made from mRNA by reverse transcriptase, used for obtaining eukaryotic genes because the DNA has introns that do not code for protein, lacks introns

contigs are

larger contiguous set of nucleotide sequences this is a computerized automated process

Subunit vaccines

made from pathogen proteins in genetically modified yeasts

Describe how a genomic library is made

made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome

Short sequences of RNA that are used in a wide variety of cell to regulate gene expression are called?

micro RNA's

A region of DNA in a plasmid that is recognized by a wide variety of restriction enzymes is call the?

multicloning site

DNA vaccines

nonpathogenic viruses carrying genes for pathogen's antigens as

These enzymes have the unique property of recognizing and clipping at base sequences?

palindromes

PCR

polymerase chain reaction this procedure rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques

The creation of customized organisms w/customized properties is called?

synthetic biology

amplicons are

the source or product of natural or artificial amplification or replication events

metagenomics

the study of genetic material directly from environmental samples

PCR CYCLE 2

the two strands resulting from the first cycle now each serve as templates a the same three steps occur. each subsequent cycle converts the new DNAs to amplicons and doubles the number of copies

List at least five applications of DNA technology.

therapeutic applications; gene silencing genome projects; shotgun sequencing human genome project; humane proteome project scientific application

gene therapy

to replace a defective or missing gene

Ligase is necessary for?

to seal the sticky ends together by rejoing the phosphate -sugar bonds cut by endonucleases

Bioinformatics

understanding gene function via computer- assisted analysis

fluorescent in situ hybridization (FISH) is

when probes are applied to intact cells and observed microscopically for the presence and location of specific genetic marker sequences on genes.

Microarrays are used to monitor?

which genes are being expressed