Genetic Engineering

What is after the Sanger's method

Dye-labeled segments of DNA, copied from template with unknown sequence. Dye-labeled segments are aplied to a capillary gel and subjected to electrophoresis. Computer-generated result after badns migrate past detector.

What are the new plasmid called

recombinant DNA plasmids

What is the name of the techniques used to make insulin?

recombinant DNA technology

What is the purpose of recombinant DNA in crop plants

the gene gives resistance to specific herbicides, diseases and pests.

Gel electrophoresis of DNA fragments of different size

1) DNA restriction fragments are placed in the well of an agrose or polyacrylamide gel. 2) apply electric field. fragments will move from the negative pole (anode) to the positive pole (cathode). 3) subject to autoradiography or incubate with fluorescent dye. Signal corresponding to DNA banding

Process of PCR

1) incubate target DNA at 94ºC for a minute to separate the strands. 2) add primers, nucleotides (deoxynucleotides), and DNA polymerase. Short oligos of 15-20 nucleotides bind to DNA sequence we want to amplify 3) primers attach to single-stranded DNA during incubation at 60ºC for 1 minute. 4) Incubate at 72ºC for 1 minute. During this time, two copies of target DNA are formed. 5) Repeat the cycle of heating and cooling 20-30 times

Six basic steps in a recombinant DNA experiment.

1) preparation (isolation) of DNA with gene of interest and vector. 2) Cleavage of DNA at particular sequences (restriction site) by restriction enzymes. Insert DNA can be added at specific points that have been cleaved. 3) ligation of DNA fragments in the plasmid (vector). Joining of the fragments. 4) Introduction of recombinant DNA into compatible host cells. Genetic transformation is the uptake of foreign DNA by a host cell. 5) Replication and expression of recombinant DNA in host cells. Cloning vectors allow insert DNA to be replicated in host cells. 6) Identification of host cells that contain recombinant DNA of interest. Screening a large number of DNA clones for desired fragment.

How are transferred DNA fragment joined to plasmid

2 linear DNA cut by the same restriction enzyme are joined by DNA ligase

Calculate how many fragments are created from PCR

2*n n = number of repeated cycles.

Where are DNA segments transferred into in E.coli

A plasmid (vector)

Why don't need to use SDS (chemical reagents) before gel electrophoresis of DNA

All DNA molecules are negative charged due to the phosphate backbone.

Example of recombinant DNA molecule created in nature

Bacteriophage or eukaryotic virus infects a host cell and integrates its DNA into the host creating a recombinant DNA molecule.

Applications of PCR

Clone DNA for recombination. Amplify DNA to detectable levels. Sequence DNA. Diagnose genetic disease. Detect pathogens.

What would you do if you need to analyze the DNA from a crime scene?

Collect DNA samples from the crime scene, and the suspects.

What are some of the genetic engineering methods

Cut DNA into segments using restriction enzymes. Gel electrophoresis to separate segments

Why must gel electrophoresis go through autoradiography or be incubated with fluorescent dye

DNA fragments cannot be seen without fluorescent dye. chemical can be inserted into double helical structure of DNA.

What to do with collected DNA from crime scene and suspect

Extract DNA. Cut and/ or amplify the DNA samples (restriction fragments)

DNA sequencing using the Sanger method

Four separate polymerization reactions are performed using ddATP, ddGTP, ddTTP, ddCTP. Low concentration of ddNTP used for each reaction (and 1 ddNTP in each sample)

How to analyze the DNA collected in forensics science?

Gel electrophoresis of DNA 1) load DNA into wells. 2) Turn on power supply. 3) Watch fragments travel through gel. 4) Observe separated fragments.

Humulin production process

In huge vats (fermentation tank) to produce large amount of bacteria. liquid culture of bacteria. Bacteria contain insulin gene. Insulin is produced. Insulin collected, purified, tested for quality and packaged.

Expression of Proteins using recombinant DNA technology

Many proteins are normally expressed at very low concentrations within cells, which makes isolation of sufficient amounts for analysis difficult. To overcome this problem, DNA expression vectors can be used to produce large amounts of full length proteins.

How are molecules traveled in gel electrophoresis

Molecules move through pores in gel at a rate inversely proportional to their chain length. smaller fragments easier to travel through the gel. Intensity of the band indicate concentration of the DNA fragment

What must be done when using E.coli

Need to eliminate endotoxin from products.

What new capability does E. coli with recombinant DNA plasmids have?

Produces a 'new' protein from that gene segment.

What is done with the recombinant DNA plasmids

Put back into E. coli. E. coli continues living and divides.

Making Recombinant DNA

Recombinant DNA molecules are constructed with DNA from different sources. Recombinant DNA molecules are created often in nature.

Gel electrophoresis of bacteriophage lambda

Restriction digest of bacteriophage lambda. Four restriction enzymes used. Sizing gel sepeartes fragments (smallest move fastest)

What is the method of sequencing DNA using dideoxynucleotides

Sanger method

What is Sanger method

Sanger method uses 2', 3'-dideoxynucleotide triphosphates (ddNTPs) which are incorporated at the 3' end of a growing DNA chain in place of a dNTP. Elongation terminated: since ddNTPs lack a 3'-hydroxyl group (H instead of OH), subsequent nucleotide addition cannot take place. Small amounts of ddNTP's terminate replication of some chains at each step, leaving a set of fragments of different lengths. Extension of DNA fragment terminate at random position.

What do plasmids have

Strong promoter. ribosome-binding site. multiple cloning site. transcription termination signal. marker gene. origin of replication.

What is the purpose of the polymerase chain reaction (PCR)

amplify selected DNA sequences. make multiple copies of a piece of DNA enzymatically

What are examples of protein product of gene

amylase, cellulase, and other enzymes prepare fabrics for clothing manufacture. human growth hormone treats stunted growth.

Where else is recombinant DNA used

To introduce a gene into crop plants.

Why use E.coli in genetic engineering

Used because it is easily grown and its genomics is known.

What is the advantage of Flavr Savr tomato

a marketing advantage. It can sit on the shelf longer. It can be picked at a later stage of ripeness.

What is genetic engineering

a set of methods

What is stone washed denim look

achieved by genetic engineering. clone cellulase genes in bacteria to produce large quantities of cellulase. Sell to textile manufacturers. Enzyme break down cotton fibres

Why use agrose form gel-type matrix instead of polyacrylamide

agrose gel is semi-solid. DNA larger than protein. smaller pore size in polyacrylamide, harder for DNA to travel through. therefore agrose gel is more suitable.

What may be used to dye DNA fragments

astrium bromide. But it is a strong carcinogen (cause mutation in DNA)

What do vectors have

at least one unique cloning site: a sequence cut by a restriction endonuclease to allow site-specific insertion of foreign DNA.

What are marker genes for

carry genes conferring antibiotic resistance. allow detection of cells. Bacterial plasmid vectors can carry a B-lactamase marker gene.

How to get product from E.coli

cells must be lysed to get product.

Before modern biotechnology, where did insulin come from?

cows, pigs, horses.

What are examples of gene cloned

gene for pest resistance inserted into plants. gene alters bacteria for cleaning up toxic waste

What is the Flavr Savr tomato

genetically engineered tomato to reduce rate of spoilage.

What is melatonin

hormone to adjust our biological clock.

What is the function of B-lactamase

hydroylze B-lactam antibiotics (e.g. ampicillin).

What was the world's first genetically engineered pharmaceutical product?

insulin

Why is the genetically engineered insulin better?

it is made from a human gene. Does not cause allergic reactions. ample supply available. Affordable, safe.

Selection strategies using marker genes

only cells transformed with plasmids expressing the B-lactamase gene are ampicillin resistant and can grow in media containing ampicillin (ampR)

What are expression vectors

plasmids that have been engineered to contain regulatory sequences for transcription and translation. Eukaryotic genes can be expressed in prokaryotes

What can be a cloning vector

plasmids, bacteriophages, viruses, small artificial chromosomes.

What is used to cut DNA into segments

restriction enzymes

What are the crop plants that uses recombinant DNA technology

rice (golden rice) soybeans. cotton

What is the advantage of recombinant melatonin

safer than natural melatonin because melatonin is secreted from cow brain. Recombinant melatonin free from contamination.

What are plasmids

small, circular DNA molecules used as vectors for DNA fragments to 20kb. Replicate autonomously within a host cell. Can carry small to medium fragments.

What are the features of cloning vectors

some vectors can be replicated autonomously in a host cell, other vectors can be integrated into the host chromosome.

What are examples of sequences using expression vectors

strong promoters, ribosome-binding site, transcription terminators.

What is SDS

to break non-bonding forces of proteins. to apply negative charges on protein (make all protein (-) charge). Enable fragments to be separated based on charge.

What are the goals of recombinant DNA

to make copies of gene. to make protein product of gene.

What is gel electrophoresis for?

to separate segments

What is done to separated segments

transfered to another organism.

What is the purpose of genetic engineering/ recombinant DNA technology

used to study and manipulate genetic material.