Biotechnological ToolsBiotechnological Tools

What are we doing here?!?!What are we doing here?!?!

One of the major advances in genetic research is the usage One of the major advances in genetic research is the usage of recombinant DNA.of recombinant DNA.Recombinant DNARecombinant DNA refers to the practice of placing DNA refers to the practice of placing DNA (genes) from one organism into the genome of a second (genes) from one organism into the genome of a second organism in hopes that the second organism will make use organism in hopes that the second organism will make use of the genes and make the proteins for which the genes of the genes and make the proteins for which the genes encode.encode.Basically, you take a desired gene from one thing and stick Basically, you take a desired gene from one thing and stick it into another so it too will be able to make the desired it into another so it too will be able to make the desired protein from the inserted gene.protein from the inserted gene.

To understand and perform any biotech work you have to To understand and perform any biotech work you have to know the tools and the procedures needed in order to get know the tools and the procedures needed in order to get the job done. So let’s take a look…the job done. So let’s take a look…

Tools of the TradeTools of the Trade

Restriction EndonucleasesRestriction EndonucleasesAKA – Restriction EnzymesAKA – Restriction EnzymesThese enzymes cut DNA at specific sequences called These enzymes cut DNA at specific sequences called recognition sitesrecognition sites..The cuts at the recognition sites can be in the form of The cuts at the recognition sites can be in the form of either blunt ends or sticky ends (with sticky ends being either blunt ends or sticky ends (with sticky ends being the more useful of the two).the more useful of the two).Restriction enzymes naturally occur in bacteria as a Restriction enzymes naturally occur in bacteria as a defense against viral infection. The virus would inject its defense against viral infection. The virus would inject its DNA into the bacterial cell only to be cut into many DNA into the bacterial cell only to be cut into many pieces and be rendered useless.pieces and be rendered useless.

Sticky vs. Blunt EndsSticky vs. Blunt EndsThere are two possibilities when a restriction There are two possibilities when a restriction enzyme cuts through the DNA.enzyme cuts through the DNA.

1.1. Sticky EndsSticky Ends – Staggered ends on a DNA molecule with – Staggered ends on a DNA molecule with short, single-stranded overhangs.short, single-stranded overhangs.

2.2. Blunt EndsBlunt Ends – A straight cut, down through the DNA that – A straight cut, down through the DNA that results in a flat pair of bases on the ends of the DNA.results in a flat pair of bases on the ends of the DNA.

Sticky ends are preferred in the lab because the Sticky ends are preferred in the lab because the single-stranded overhangs are complementary to single-stranded overhangs are complementary to each other and can be stuck back together with each other and can be stuck back together with other sticky ends made from the same restriction other sticky ends made from the same restriction enzyme.enzyme.Blunts ends can match up with any other blunt end Blunts ends can match up with any other blunt end so you don’t get that exact match between the two so you don’t get that exact match between the two pieces of DNA you are trying to put together.pieces of DNA you are trying to put together.

Sticky Ends vs. Blunt EndsSticky Ends vs. Blunt Ends

Methylases & DNA LigaseMethylases & DNA Ligase

MethylasesMethylases are enzymes that add a methyl group are enzymes that add a methyl group (–CH(–CH33) to a nucleotide in the recognition site of the DNA.) to a nucleotide in the recognition site of the DNA.

This extra methyl on the nucleotide changes the shape This extra methyl on the nucleotide changes the shape of the recognition site and the restriction enzyme is of the recognition site and the restriction enzyme is unable to cut it because of the change in shape.unable to cut it because of the change in shape.Methylases are also naturally found within bacteria – it is Methylases are also naturally found within bacteria – it is how they protect their own DNA from their restriction how they protect their own DNA from their restriction enzymes.enzymes.

DNA LigaseDNA Ligase reforms the phosphodiester bonds between reforms the phosphodiester bonds between adjacent nucleotides when you are trying to connect the adjacent nucleotides when you are trying to connect the foreign DNA fragment and the host cells DNA.foreign DNA fragment and the host cells DNA.

Gel ElectrophoresisGel ElectrophoresisGel electrophoresisGel electrophoresis is a technology that separates is a technology that separates molecules based on charge and size by sorting them molecules based on charge and size by sorting them in a gel meshwork.in a gel meshwork.The gel (meshwork) is like the rubber band tunnel at The gel (meshwork) is like the rubber band tunnel at McDonald’s. You know the one – that thin, long, trap McDonald’s. You know the one – that thin, long, trap with those thick black bands running through it this with those thick black bands running through it this way and that. You have to really squirm your way way and that. You have to really squirm your way through it to get to the other side.through it to get to the other side.Well…The smaller you are the easier it is to fly Well…The smaller you are the easier it is to fly through it. And vice-versa of course.through it. And vice-versa of course.The charge on the molecule dictates the direction the The charge on the molecule dictates the direction the molecule moves within the gel. Opposite charges molecule moves within the gel. Opposite charges attract so positive molecules move to the negative attract so positive molecules move to the negative electrode while negative molecules move to the electrode while negative molecules move to the positive electrode.positive electrode.

Tasing the DNATasing the DNADNA has a negative charge – all those phosphates in the DNA has a negative charge – all those phosphates in the backbone!backbone!We use that property to make the DNA move. An electric We use that property to make the DNA move. An electric current is passed through the gel and charged particles current is passed through the gel and charged particles (like DNA) will move in response to that electric current.(like DNA) will move in response to that electric current.Your DNA samples are loaded into Your DNA samples are loaded into wellswells (small pits) (small pits) within the gel. The wells for DNA are near the negative within the gel. The wells for DNA are near the negative electrode so they will move to the positive electrode – electrode so they will move to the positive electrode – opposites attract!opposites attract!When the switch is thrown - the DNA fragments move. When the switch is thrown - the DNA fragments move. The smaller the fragment, the further it moves within the The smaller the fragment, the further it moves within the gel.gel.After the gel has been run, it is treated with After the gel has been run, it is treated with ethidium ethidium bromidebromide that clings to rungs of the DNA and causes it to that clings to rungs of the DNA and causes it to fluoresce (glow) under a UV light.fluoresce (glow) under a UV light.

Gel ElectrophoresisGel Electrophoresis

PlasmidsPlasmidsPlasmidsPlasmids are small, circular pieces of DNA that can exit are small, circular pieces of DNA that can exit and enter bacterial cells.and enter bacterial cells.They contain “bonus” DNA in that they can have genes in They contain “bonus” DNA in that they can have genes in them that allow the bacterial cell to become resistant to them that allow the bacterial cell to become resistant to some of the things that would normally kill it. These genes some of the things that would normally kill it. These genes are known as are known as resistance genesresistance genes..We can insert foreign DNA into plasmids and put them We can insert foreign DNA into plasmids and put them into bacterial cells for them to use.into bacterial cells for them to use.We use the resistance genes to show us whether or not a We use the resistance genes to show us whether or not a plasmid has successfully adopted a plasmid that contains plasmid has successfully adopted a plasmid that contains the foreign DNA. The foreign DNA interrupts one of the the foreign DNA. The foreign DNA interrupts one of the resistance genes on the plasmid and the bacteria that has resistance genes on the plasmid and the bacteria that has that plasmid loses its ability to be resistant to whatever that plasmid loses its ability to be resistant to whatever substance it provide the resistance against.substance it provide the resistance against.

Plasmid PicsPlasmid Pics

TransformationTransformation

The introduction of foreign DNA into a bacterial The introduction of foreign DNA into a bacterial cell is known as cell is known as transformationtransformation..

Transformation requires a Transformation requires a vectorvector (or delivery (or delivery vehicle) that will bring the foreign inside the vehicle) that will bring the foreign inside the bacterial cells. Plasmids or viruses are normal bacterial cells. Plasmids or viruses are normal vectors for transformation.vectors for transformation.

Any cell that has been successfully transformed Any cell that has been successfully transformed and that now contains the foreign DNA is known and that now contains the foreign DNA is known as a as a competent cellcompetent cell..

To Sum It All Up…To Sum It All Up…

These are the basic These are the basic tools needed to tools needed to perform any perform any experiments or work experiments or work in a biotech lab.in a biotech lab.Future lectures will Future lectures will deal with some of the deal with some of the techniques and techniques and processes that are processes that are employed in these employed in these same labs.same labs.

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