Molecular Cloning

Views:
 
     
 

Presentation Description

An introductory lecture on molecular cloning as part of a genetics class. Covers amplification of genes in E. Coli and PCR.

Comments

By: eldebaky (48 month(s) ago)

it is very good ppt and it is really useful to me in molecular biology and cloning study. thanks

Presentation Transcript

Molecular Cloning : 

Molecular Cloning Dr. Robin Herlands Genetics, Biology 300, Nevada State College Created Fall 2008 with some images from Hartwell, 3rd Ed. “Genes to Genomes”

Slide 2: 

In each of your cells, there are 20-30,000 genes. 250,000,000 base pairs Learning about genes has required scientists to develop tools to manipulate the genome

Slide 3: 

Being able to simply: Cut DNA into pieces Ligate different pieces together Visualize DNA fragment size differences Has allowed us to MAP genes and the genome Compare species/organisms Discover mutations leading to disease

Fragmenting DNA : 

Fragmenting DNA DNA is fragmented into smaller, more maneagable pieces with restriction enzymes Restriction enzymes cut DNA phosphate-sugar backbone at a very particular sequence. Hundreds of specific enzymes are now commercially available - just add to DNA and wait an hour! Cutting DNA in this manner is called digestion.

Slide 5: 

Restriction Enzymes are very specific. Each will cut at a particular 4-6bp sequence. Ex: EcoRI binds and cuts GAATTC

Slide 6: 

Restriction Enzymes can cut DNA in a few different ways Fig. 9.2

Slide 7: 

“Sticky ends” means that the overhangs want to bind to a complementary sequence. If original cut DNA is left alone, it will re-assemble. If digested DNA from another source is added, with identical sticky ends, it can be ligated with the original DNA to create an intentional insertion.

Slide 8: 

http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/bx15_01.jpg

Fragment Sizes Depend on DNA Sequence, but can be estimated. : 

Fragment Sizes Depend on DNA Sequence, but can be estimated. If Rsa1 recognizes a 4 bp sequence, it will cut on average once every 44 Avg. fragment = 256bp Is Not1 recognizes a 8 bp sequence, it will cut on average once every 48 Avg. fragment = 6500 bp

How do you learn something from these fragments? : 

How do you learn something from these fragments? If the size of the fragments can be determined, a kind of map can be made of the genome. If the sequence of the fragments can be determined, a complete, sequenced genomic map could be made.

Determining the Size of DNA Fragments : 

Determining the Size of DNA Fragments DNA size is determined by gel electrophoresis Electrophoresis is the movement of charged molecules in an electric field.

Slide 16: 

If a gene has a known sequence, using restriction enzymes can help detect mutations in a gene. 1000 500 400 300 200 100

Slide 17: 

1000 500 400 300 200 100

Slide 18: 

If a gene sequence is unknown, the fragments can be sequenced (shown later)… or a basic map of the gene can be made based on restriction enzyme sites.

Slide 19: 

DNA is divided into portions Portion 1 is treated with an R.E. Portion 2 is treated with a 2nd R.E. Portion 3 is treated with both enzymes.

Slide 20: 

Digested DNA is loaded onto an electrophoresis gel and run.

Slide 21: 

Through process of elimination, the results are mapped

Cloning : 

Cloning What does cloning mean to you? Let’s try to come up with a good definition.

Slide 24: 

Dolly with her baby: http://www.sciencenewsforkids.org/articles/20040128/a299_1494.jpg

Slide 25: 

Cloning Dolly pic: http://static.howstuffworks.com/gif/cloning-sheep.gif

Slide 26: 

Cloning : process of producing populations of genetically-identical individuals. Molecular Cloning : processes used to create copies of DNA fragments Cellular Cloning : making genetically identical cells Main idea: Production of multiple copies of something

Slide 27: 

Does anyone know how molecular cloning works?

DNA Cloning : 2 methods : 

DNA Cloning : 2 methods Molecular cloning using a live cell for amplification Polymerase chain reaction (PCR) - an amplification process which requires no live cells.

Molecular cloning with live cell intermediate: Summary : 

Molecular cloning with live cell intermediate: Summary First you need to isolate the DNA you want to clone, usually a gene You put this DNA into a vector, a temporary small circular piece of DNA This DNA is then provided to particular bacteria which can uptake the DNA into their own cells Bacteria duplicate the DNA as they divide: grow lots of bacteria, grow lots of DNA DNA is re-isolated from the bacteria.

Slide 30: 

Break?

Molecular cloning with live cell intermediate: Summary : 

Molecular cloning with live cell intermediate: Summary First you need to isolate the DNA you want to clone, usually a gene You put this DNA into a vector, a temporary small circular piece of DNA This DNA is then provided to particular bacteria which can uptake the DNA into their own cells Bacteria duplicate the DNA as they divide: grow lots of bacteria, grow lots of DNA DNA is re-isolated from the bacteria.

Step 1: : 

Step 1: Restriction fragments are generated using R.E. digestion The vector DNA must also be digested to have identical sticky ends The two digestions are mixed together along with DNA Ligase. Remember what DNA Ligase does?

Vectors : 

Vectors There are several types of vectors. Most commonly used is a bacterial plasmid: Circular DNA commonly found in bacteria, does not contain any bacterial chromosomal DNA. New technology has allowed us to make really creative plasmids…

Slide 35: 

This plasmid contains an Origin of Replication, several R.E. sites, and some other special genes that will allow scientists to ascertain if cloning occurred successfully. http://www.sciencesoftware.com/ProductImagesA/circular_plasmid_maps.gif

Slide 36: 

For example, plasmids can contain selctable markers, such as an antibiotic resistance gene (AmpR = ampicillin resistance gene) That way, scientists can add antibiotic to the bacterial growth media and only bacterial cells that have taken-up the plasmid will continue to grow.

Slide 37: 

Plasmid with AmpR picture

Slide 38: 

Step 1 Review: Digest DNA to get your gene/DNA of interest Digest vector Ligate your DNA fragment and the vector to create a recombinant vector or recombinant plasmid.

Step 2: Live Cells Take Up the Recombinant Vector : 

Step 2: Live Cells Take Up the Recombinant Vector Certain types of bacteria are able to take up pieces of DNA from the external environment. Remember the Griffith Expt? Bacteria that can do this are called competent bacteria. The process of a bacteria taking up new DNA from the outside is called transformation.

Slide 41: 

When you made your recombinant plasmid, not all plasmids likely took up your particular DNA. How can you distinguish bacteria that contain YOUR recombinant plasmid from those that contain empty plasmid (both will be Amp resistant)?

Blue White Screen : 

Blue White Screen Common technique : include in plasmid a gene, lacZ, that encodes for beta-galactosidase This enzyme can convert a substrate, X-gal, into a visibly blue product. When you insert your DNA into the plasmid, have it disrupt this lacZ gene. Blue colonies represent bacteria that have the empty plasmid. White colonies represent bacteria that you the recombinant plasmid.

Slide 44: 

Blue white real picture http://upload.wikimedia.org/wikipedia/en/thumb/6/66/Blue_White_Screen2.JPG/250px-Blue_White_Screen2.JPG

Amplifying your DNA even more : 

Amplifying your DNA even more You “pick” a white colony to “grow up” Toothpick to colony, drop in a flask of media, grow at body temperature until there are tons of bacteria.

Recovering your DNA : 

Recovering your DNA You now want to retreive your amplified (cloned) DNA. Lyse the cells with a chemical reagent, extract DNA Plasmid DNA can be separated from bacterial DNA due to the size difference between them. Gel Electrophoresis CsCl solution gradient with centrifugation

Recovering your DNA -2 : 

Recovering your DNA -2 Cut plasmid DNA with restriction enzymes to remove your segment. Run on gel electrophoresis once more to separate the plasmid DNA from your restriction fragment.

2nd Method of Cloning: PCR : 

2nd Method of Cloning: PCR Polymerase Chain Reaction is an exponential amplification of a piece of DNA without a live cell develop short oligonucleotides (~ 20bp) called primers which are complementary to sequences flanking the target DNA. similar to RNA primers used in DNA replication.

Oligonucleotide primers bind DNA : 

Oligonucleotide primers bind DNA

Slide 50: 

A polymerase and free nucleotides are added to the mixture of DNA and primers. A temperature change cause original DNA helix to unwind The polymerase makes complementary strands of the now separate strands. The temperature is lowered, primers bind to new DNA strands, process is repeated.

Each cycle of replication doubles amount of target DNA. : 

Each cycle of replication doubles amount of target DNA. 1 2 3

Exponential amplification : 

Exponential amplification

What do you do with cloned DNA? : 

What do you do with cloned DNA? Send it away for sequencing to look for mutations, build a DNA library to map the genome, or try to better understand the protein it will make. Use it for a genetic engineering project - perhaps you needed lots of this gene because you’re going to add it to a viral vaccine or make a transgenic mouse.

Therapuetic Application : 

Therapuetic Application Special plasmids called “expression vectors” can be used to induce the bacteria to make the protein of your target DNA. Rather than just make TONS of copies of the DNA, you can get the bacteria to make TONS of protein. How might this be useful?

Slide 56: 

Some diabetics cannot make insulin, and have to inject insulin daily. We now use bacteria, cloning technology, and expression vectors to grow up insulin for commercial use. Hemopheila, PKU, and several of the diseases we discussed were loss-of-function mutations. Growing up protein this way is inexpensive and fast and can save lives!