Module 2- Biotechnology

Plasmid DNA

small circular pieces of DNA found primarily in bacteria

1 to 4 kb

How small is Plasmid DNA?

Vectors

Pieces of DNA that can accept, carry, and replicate other pieces of DNA.

Calcium Chloride Transformation of Bacterial Cells

A process for inserting foreign DNA into bacteria. (ineffective)

What is the 1st step?

Treat bacterial cells with calcium chloride while chilled on ice for 30 minutes.

What is the 2nd step?

Add plasmid DNA to cells chilled on ice.

What is the 3rd step?

Heat the cell and DNA mixture (30 seconds at 42 degrees Celsius)

What is the 4th step?

Plasmid DNA enters bacterial cells and is replicated and express their genes.

Electroporation

applies a brief pulse of high voltage electricity that creates tiny holes in the cell wall that allows DNA to enter.

What are advantages of electroporation?

Rapid, requires few cells, can be use to introduce DNA into other cell types, more efficient process.

Selection

process that facilitates the identification of recombinant bacteria from bacteria that contain plasmid without foreign DNA, while preventing the growth of non-transformed bacteria.

Antibiotic selection

transformed cells are cultured on plates containing antibiotics to identify recombinant bacteria and non-transformed bacteria

What does Antibiotic selection NOT select?

plasmid containing foreign DNA vs. recircularized plasmid with no DNA insert.

Blue-white selection with X-gal in media

DNA is cloned into plasmid restriction site within LacZ gene.

What happens when Blue-white selection is interrupted by an inserted gene?

the laxZ gene cannot produce functional Beta galactosidase.

What is non-functional lacZ?

White colony. Clones. Genetically identical bacterial cells each containing copies of recombinant plasmid.

What happens when X-gal is added to media in petri plate?

functional lacZ=blue colony=clones= genetically identical bacterial cells each containing recircularized plasmid. no transgene.

What was the first recombinant human protein marketed?

Insulin, 1982

What was the next human protein marketed?

growth hormone, 1985

What was source of growth hormone prior to recombinant technology?

cadavers

What makes a good vector in regards to size?

small enough to be separated from chromosomal DNA of host plasmid

What makes a good vector in regards to origin of replication?

site for DNA replication that allow plasmids to replicate independently from host chromosome

What is copy number?

number of plasmids in the cell; normally small (recombinant plasmids have high copy numbers)

What makes a good vector in regards to Multiple cloning site?

recognition sites for several restriction enzymes in which DNA insert is cloned into

What makes a good vector in regards to Selectable marker genes?

allow to select for transformed colonies

What makes a good vector in regards to RNA polymerase promoter sequences ?

used for transcription in vitro and in vivo

What prevents restriction enzymes produced by a bacteria from digesting its own genome?

Methylated DNA

DNA Libraries

Collections of cloned DNA fragments from a particular organism contained in plasmid vectors within host bacteria (ex. E. colt

What are the two types of libraries?

Genomic DNA libraries and Complementary DNA libraries (cDNA libraries)

Genomic Libraries: What happens to Chromosomal DNA?

isolated & digested with a restriction enzyme which produces many fragments that include the entire genome

Genomic Libraries: What happens to the vector (plasmid)?

digested with same enzyme

Genomic Libraries: What happens to DNA ligase?

used to ligate chromosomal DNA fragment within vector DNA (plasmids)

Genomic Libraries: What happens to recombinant vectors?

used to transform bacteria, and theoretically each bacteria will contain a single recombinant plasmid

What is the 1st disadvantage of genomic libraries?

Introns are cloned in addition to exons. Majority of the library will contain non-coding pieces of DNA.

What is the 2nd disadvantage of genomic libraries?

Many organisms have large genomes, searching for the gene of interest is time consuming.

What is the 3rd disadvantage of genomic libraries?

Does not provide information on levels of gene expression.

cDNA libraries

mRNA from tissue of interest is extracted.

cDNA libraries need to make double stranded DNA from mRNA: How?

Short linker double stranded DNA sequences, which contain restriction enzyme recognition sites, are added to the ends of the cDNA.

Cut with restriction enzyme, cut vector with same enzyme, ligate fragments to create recombinant vectors

Then transform bacteria with recombinant vectors

Advantage 1 of cDNA libraries over genomic libraries

Collection of actively expressed genes in the cells or tissues from which the mRNA was isolated

Advantage 2 of cDNA libraries over genomic libraries

Introns are NOT cloned

Advantage 3 of cDNA libraries over genomic libraries

Can be created and screened to isolate genes that are primarily expressed only under certain conditions in a tissue

Disadvantage of cDNA libraries

Can be difficult to make the cDNA library if a source tissue with an abundant amount of mRNA for the gene is not available

colony hybridization

library screening to identify the gene of interest.

colony hybridization 1st step

Bacteria with recombinant DNA grown on agar plate

colony hybridization 2nd step

Nylon or nitrocellulose filter is placed over the plate

colony hybridization 3rd step

Treat filter with alkaline solution to lyse the cells and denature the DNA; then bake filter or UV exposure

colony hybridization 4th step

Denatured DNA binds to filter as single-stranded DNA

colony hybridization 5th step

Filter is incubated with a probe that is tagged with a radioactive nucleotide or fluorescent dye

colony hybridization 6th step

Probe binds by hydrogen bonding to complementary sequences on the filter = hybridization

colony hybridization 7th step

Filter is washed to remove excess unbound probe

colony hybridization 8th step

Filter exposed to X-rav film or digital camera to detect fluorescent probe

fluorescent probe 9th step

Film or picture is then compared to the original agar

plate to identify which colonies contained recombinant

plasmid with the gene of interest

What does the type of probe depend on?

What is already known about the gene of interest. Ex: rat probe to screen human library.

Does library screening result in the cloning of the full-length gene?

no. Usually get small pieces of the gene: the pieces are sequenced and scientists look for overlapping sequences.

What is looked for to know when the full length of the gene obtained?

start and stop codons.

What is polymerase chain reaction?

Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time

When was polymerase chain reaction developed and by who?

1983 by Kary Mullis polymerase chain reaction (PCR)

PCR 1st step

Target DNA to be amplified is added to a tube,mixed with nucleotides (dATP, dCTP, dGTP, dTTP)., buffer with MgCl2, and DNA polymerase.

PCR 2nd step

Paired set of Forward and Reverse Primers are added - short single-stranded DNA oligonucleotides (18-22 nucleotides long)

PCR 3rd step

Reaction tube is placed in an instrument called a thermocycler

What does the thermocycler take the DNA through?

through a series of reactions called a PCR cycle

What are the 3 cycles of PCR cycle?

Denaturation, Annealing, and extension.

What happens in denaturation?

heat to 94 °C to 96 °C

What happens during annealing?

(hybridization) - in which primers H bond with complementary bases at the opposite ends of target sequence at 52 °C to 58 °C

What happens during extension?

(elongation) - DNA Pol copies target

DNA at 70 to 75 °C

What happens at the end of PCR cycle?

the amount of DNA has doubled

How many times are cycles repeated?

20-30 times

Primer design with a loop?

Harpin loop

Primer design with same color?

homodimer

Primer design with different color?

heterodimer

Advantage 1 of PCR

amplify millions of copies of target DNA from small amount of starting material in short period of time

Advantage 2 of PCR

To calculate the number of copies of target DNA starting with 1 molecule of DNA use this equation 2N in which N represents number of PCR cycles

Assume you want to do 2 PCR cycles to amplify your DNA insert, how many copies of DNA will you have at the end of your PCR?

2 to the power of 2. 4.

What is Taq DNA polymerase?

isolated from a species known as Thermus aquaticus that thrives in hot springs

Applications of DNA polymerase?

- Making DNA probes

- Studying gene expression

- Detection of viral and bacterial infections

-Diagnosis of genetic conditions

-Detection of trace amounts of DNA from tissue found at crime scene

- Detection of DNA from fossilized tissue

Cloning PCR products is more effective than what?

DNA libraries.

What is the disadvantage of cloning PCR?

Need to know something about the DNA sequence that flanks the gene of interest to design primers

What does Taq polymerase do in PCR?

polymerase puts a single adenine nucleotide on the 3' end of all PCR products

What knowledge is used to the researchers' advantage?

T vector that has single stranded thymine on each end so can complementary base pair with the adenine in the PC products

What is the chain termination sequencing?

Sanger method. Important to determine the sequence of nucleotides of the cloned gene

When was the chain termination sequencing developed and by who?

Frederick Sanger & colleagues in 1977

The sanger method requires?

-single primer annealing to denatured DNA template

- all 4 dNTPs

-DNA Polymerase

-dideoxynucleotide (ddNTP) which has a 3' H instead of 3'OH on the deoxyribose so it cannot form a phosphodiester bond with the incoming nucleotide & so gets terminated

Original Sanger Method 1st step

Had four separate reaction tubes and each contained the same DNA; radioactively labeled single primer; all 4 dNTPs; and a small amount of one daNTP per tube; DNA Pol

Old Sanger Method 2nd step

Over time a ddNTP will be incorporated into all the positions in the newly synthesized strands creating fragments of varying lengths that are terminated at the ddNTP

Original Sanger Method 3rd step

fragments are separated on polyacryamide gel

Original Sanger Method 4th step

Autoradioaraphy used to then identify radioactive fragments

How is the gel read on the original sanger method?

bottom to top as individual nucleotides

How is the sequence generated using the original sanger method?

complimentary to the sequence on the template strand in the vector

How many nucleotides are sequenced per reaction during the original sanger method?

200-400 nucleotides

What did using capillary electrophoresis enable for the Sanger method? How was it helpful?

enables greater than 600 nucleotides to be sequenced per reaction. very helpful for completing human genome project.

What is the procedure of using capillary electrophoresis?

uses only 1 reaction tube instead of 4

Capillary Electrophoresis (CE)

1st step

ddNTPs are each labeled with a different fluorescent dye

Capillary Electrophoresis (CE)

2nd step

Samples are separated on a single-lane capillary gel that is scanned

with a laser beam

Capillary Electrophoresis (CE)

3rd step

Laser stimulates fluorescent dye on each DNA fragment which emits

a different wavelength of light for each different colored ddNTP