Chapter 17 : Biotechnology

What is a Recombinant DNA?

A single DNA molecule made from two different sources.

What is the key for entering a sequence of DNA into another piece of DNA?

Restriction Endonucleases

Why are Restriction Endonucleases important?

They cleave DNA at specific sites. Like a knife cutting a rope in the same spot every time.

What are two benefits of cleaving DNA at specific sites?

1. Builds restriction maps which show crucial data for working with DNA molecules.

2. Creation of recombinant DNA molecules.

Of the three restriction enzymes, which one only cleaves at specific sites?

Type II

Type II enzymes enable _________ molecules to recognize a specific DNA sequence (4-12 bases).

Recombinant

True or False: When both strands of DNA are cut, they are complimentary to each other and combine because of the restriction endonuclease EcoRI.

True.

What is the Pallindrome and what is the significance?

They are recognition sites for most type II enzymes. They read the same sequence from both strands making them complimentary.

What is the most common separation technique used when separating DNA?

Gel Electrophoresis

True or False: The Get Electrophoresis separates based on size.

True.

Why do they separate based on size?

The larger ones move slower in the gel matrix.

Which enzyme is used to join the two DNA fragments together?

DNA Ligase

Molecular _______ involves the isolation of a particular sequence of DNA.

Cloning

What is transformation?

The introduction of DNA from an outside source into a cell.

What is a vector?

Allows you to propagate DNA in a host cell.

What are the two most commonly used vectors?

Plasmids, and artificial chromosomes.

What is the common and flexible host used for molecular cloning?

Bacterium E. Coli

What are Plasmids?

small, circular extra-chromosomal DNAs that are dispensable to the bacterial cell.

What are the three components of a plasmid vector?

1. An origin of replication to allow it to be replicated in E.Coli independently of the host chromosome.

2. A selectable marker, usually antibiotic resistance. Allows for easy identification.

3. One or more unique restriction sites where foreign DNA can be added.

What are the two successful approaches for transforming Eukaryotic cells?

Injecting DNA into the pronucleus of a fertilized egg, or injecting embryonic stem cells into an embryo.

What is the most successful plasmid?

Ti (tumor-inducing) plasmid.

What does PCR do to genetic material?

It can accelerate the pace of genetic engineering by quickly creating many clones of a DNA sequence before using a plasmid.

What are the three steps of a PCR machine?

1. DNA is denatured by first heating. (separate into two strands.)

2. Temperature is cooled to allow the primers to anneal onto ends of the targeted DNA.

3. DNA heated to 72 C (Intermediate Temperature) for Taq DNA polymerase to extend primers, creating a double strand.

True or False: The heating harms the Taq Polymerase.

False.

What is a DNA library?

A collection of DNAs in a vector that taken together represent the complex mixture of DNA.

What is the simplest form of a DNA library? And what is it?

Genomic Library, and it represents the entire genome in a vector.

What is the result of not cutting the DNA to completion in a genomic library?

Not all the sites are cleaved, and the ones that are cleaved are randomized and fragmented. They are then inserted into vectors and introduced into host cells.

Where are genomic libraries usually constructed in?

BACs.

Why is a mature mRNA transcript much smaller than the Gene?

This is due to the loss of introns by splicing.

What enzyme uses the mature mRNA to make the complimentary DNA?

Reverse transcriptase

The complimentary copied strand of DNA from the mature DNA is then used as a template by which enzyme?

DNA Polymerase

What does the DNA polymerase do to the strand?

It assembles a complementary DNA strand along it producing cDNA.

So overall, the cDNA is a double stranded copy of what?

The intron-free DNA strand.

Why may newly cloned genes help with DNA libraries?

They act as probes to detect similar genes in a library.

What is Southern Plot?

DNA is fragmented, separated, denatured, then blotted with a sheet of filter paper, some DNA is now on the sheet. The filter is incubated and any DNA fragment that is complimentary to the probe's sequence is hydrolized.

What is Northern Plot?

The same as Souther Plot except for RNA, denaturation is not required, that is only for DNA.

What is Western Plot?

The same plot sequence except it is for proteins.

What does DNA fingerprinting do?

Compares DNA from several different regions of individual genomes for identification purposes.

What is 'in vitro mutagenesis?'

The ability to create mutations at any site in a cloned gene to examine their effect on function.

Why does in vitro mutagensis produce 'knockout mice?'

A known gene is inactivated (knocked out). The effect is assessed in the adult mouse.

What are embryonic stem cells?

They can develop into different adult tissues.

The use of a knockout gene is an example of what?

Reversed Genetics

What happens with Knockout genes?

A copy of a gene (clone) is used to make a mutant that is lacking in that gene. We can assess the effect on the entire organism by eliminating a single gene.

Unlike knockout mutations, _________ can target specific RNA sequences so they are degraded and not translated into protein.

RNAi

RNAi determines the function of a gene by what method?

Silencing the expression of a gene by inserting into the cells. Changes in phenotype provide info about the function of the gene that is knocked down.

True or False: RNAi does not completely turn off a gene.

True.

What was the first eukaryotic gene to be expressed in bacteria? Why was it important?

Human Insulin, it allows easy production of insulin by bacterial to be given to diabetics.

What three problems required attention to apply insulin in bacteria?

1. Eukaryotic promoters do not work in bacteria.

2. bacteria lack posttranslational modification, cannot remove introns.

3. bacteria lack posttranscriptional modification so the polypeptide could not be cleaved into two polypeptides.

What do Subunit vaccines effect?

Pathogens

How to Subunit vaccines work?

Genes encoding a protein on the surface of the pathogen are cloned to be used to produce the vaccine.

What is DNA vaccines?

One that doesn't depend on antibodies, the blood cells killer t cells attack infected cells.