Recombinant DNA Technology

Recombinant DNA Technology combines DNA from different sources – usually different species Utility: this is done to study DNA sequences to mass-produce proteins to give recipient species new characteristics as a therapy/curative for genetic disorders (‘gene therapy’) Corn damaged by corn borer and fungi “bt-corn”, with a bacterial gene Human insulin, created in bacteria

Recombinant DNA Technology A.Overview: 1. Purify DNA 2. Cut it with restriction enzymes – create a DNA ‘library’ of the fragments 3. Insert it into a vector, creating a recombinant DNA molecule 4. Insert the vector into a host cell 5. Create a population of cells (clone) that have this new DNA. 6. The DNA or protein product can be isolated and purified, or the new organisms with this cell type can be used.

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate.

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate. - Best case – know the location of the gene and can pinpoint it

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate. - Best case – know the location of the gene and can pinpoint it - know the chromosome it is on - Worst case – have to screen the entire genome.

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate. - Best case – know the location of the gene and can pinpoint it - know the chromosome it is on - Worst case – have to screen the entire genome. Three Ways: - cut up DNA with restriction enzymes and isolate gene - make copies of gene with PCR - use m-RNA to make copies of gene with reverse transcriptase

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate. - Best case – know the location of the gene and can pinpoint it - know the chromosome it is on - Worst case – have to screen the entire genome cut it with a restriction enzyme that cuts at specific sequences and leaves specific “tails” (the fewer fragments the better!!!)

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate. - Best case – know the location of the gene and can pinpoint it - know the chromosome it is on - Worst case – have to screen the entire genome cut it with a restriction enzyme that cuts at specific sequences and leaves specific “tails” (the fewer fragments the better!!!) - cut a ‘vector’ with the same restriction enzyme and ligate them with ligase…

The vector could be plasmid (for placement in bacteria)… Absorbed by inducing a state of ‘competence’ with calcium salts, and transforming the bacteria (uptake of exogenous DNA).

…or a ‘yeast artificial chromosome’ – YAC - (“yak”) for the manipulation and study of eukaryotic genes… Bacterial plasmid that has had yeast centromeres, telomeres, and replication origins inserted. Eukaryotic gene is inserted into the plasmid, then the chromosome is “linearized” with BamHI and inserted into a yeast colony. Why insert a eukarytoic gene into yeast, rather than bacteria?

…or a virus, capable of infecting other cells with their new gene.

Identify cells that have absorbed a recombinant plasmid. - grow them on an ampicillin plate – only cells that have accepted a plasmid can grow - since the fragments insert into a functional gene for lactose metabolism, cells that have accepted a recombinant plasmid will be white (no lactose metabolism). - WHICH of THESE WHITE cells absorbed the gene of interest??? (small library is better!)

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate. - Best case – know the location of the gene and can pinpoint it - know the chromosome it is on - Worst case – have to screen the entire genome cut it with a restriction enzyme that cuts at specific sequences and leaves specific “tails” (the fewer fragments the better!!!) - cut a ‘vector’ with the same restriction enzyme and ligate them with ligase. OR 2. - use the Polymerase Chain Reaction (‘PCR’) to clone DNA.

PCR (Mullis – 1993 Nobel in Chemistry) - in preparation, you have purified the DNA of interest… it may be in very small amounts – even one cell or a DNA fragment Step 1: Denaturation of DNA: Heating the sample to o C breaks the H-bonds, denaturing the DNA (separating the helices). ~ 1 min.

PCR - in preparation, you have purified the DNA of interest… it may be in very small amounts – even one cell or a DNA fragment Step 1: Denaturation of DNA: Heating the sample to o C breaks the H-bonds, denaturing the DNA (separating the helices). ~ 1 min. Step 2: Anneal Primers: The key to PCR is that you must know something about the DNA sequence… or be able to guess. You create ss-DNA primers that anneal (h-bond) to complementary sequences in DNA when it is cooled down (50-70 o C)

PCR - in preparation, you have purified the DNA of interest… it may be in very small amounts – even one cell or a DNA fragment Step 1: Denaturation of DNA: Heating the sample to o C breaks the H-bonds, denaturing the DNA (separating the helices). ~ 1 min. Step 2: Anneal Primers: The key to PCR is that you must know something about the DNA sequence… or be able to guess. You create ss-DNA primers that anneal (h-bond) to complementary sequences in DNA when it is cooled down (50-70 o C) Step 3: Extension (Polymerization): Use a thermally stable polymerase (Taq) to polymerize DNA. One Cycle (2-5 min.)

PCR 4. Repeat Cycle: Heat up and denature, anneal, polymerize 3 hrs, ~ 25 cycles = 2 25 copies

PCR Benefits: - with correct primer, it is very sensitive and amplifies very specific sequences from very small samples - genetic testing - forensics - molecular paleontology

PCR Benefits: - with correct primer, it is very sensitive and amplifies very specific sequences from very small samples - genetic testing - forensics - molecular paleontology - primers can be used as PROBES that recognize sequences differing by single bases (alleles)

Recombinant DNA Technology A.Overview: B.Creating a DNA Library - the more you know about the location of your gene of interest, the better; the LESS DNA you will have to manipulate. - Best case – know the location of the gene and can pinpoint it - know the chromosome it is on - Worst case – have to screen the entire genome cut it with a restriction enzyme that cuts at specific sequences and leaves specific “tails” (the fewer fragments the better!!!) - cut a ‘vector’ with the same restriction enzyme and ligate them with ligase. OR 2. - use the Polymerase Chain Reaction (‘PCR’) to clone DNA. OR 3. - create c-DNA (‘complementary DNA’) from the isolated m-RNA transcript, using reverse transcriptase.

Isolate m-RNA’s in eukaryotic cell. Use a “poly-T primer” to recognize the Poly-A tails of the m-RNA. Use reverse transcriptase to polymerize the DNA.

Partially digest with Rnase. Add DNA Polmerase I (repair), which cuts out rest of RNA and polymerizes DNA across template. Add ligase to complete ds-DNA

You can also use “reverse transcription PCR” to amplify a particular c-RNA directly.

Benefits of a c-DNA Library: 1. The absence of introns means that vectors and bacteria can handle the size and structure of the eukaryotic c-DNA gene. 2.If you can localize the cell that is producing the protein of interest, then the library will only contain DNA of active (translated) genes – not ALL genes like in a whole genome library. 3. If made from m-RNA, you can amplify genes that are very low in productivity, and can amplify genes at different times of development and get a picture of gene activation.

Recombinant DNA Technology A.Overview: B.Creating a DNA Library

Recombinant DNA Technology A.Overview: B.Creating a DNA Library C. Recover the clone of interest The white cells have absorbed recombinant plasmids… but which one has absorbed the fragment we want – the one with our gene of interest?

Probes are short sequences of DNA that will bind to the gene of interest. They are either radioactive or colorimetric. Often the probe is a stretch of c-DNA.