13 - Recombinant DNA

How can we tell which new cells contain the gene of interest?

*Not all of the recombined plasmids have the gene of interest, so when you grow the new cells with the new plasmids in them, only some have the gene of interest. **Not only that, but transformation is not a very efficient process, so most bacteria mixed with the plasmids didn't even take up a plasmid successfully. How to only let the ones with the gene you are interested in grow? Make it resistant to antibiotics and grow cells in an antibiotic environment. **pBR322 has two genes for drug resistance, and the EcoR1 cutting site is between them, so once recombined, both genes would still work. However, if we use a different cutting site in the middle of one a drug resistant gene, any cell that successfully took on a recombined plasmid would LOSE its drug resistance because the recombination would have ruined those genes. So this would be a good way to discern which cells did have the recombined plasmid.

In light of those two problems, how do we identify those cells that have retained the plasmid, and give them an advantage?

*With a selectable marker—typically antibiotic resistance. If the plasmid gives antibiotic resistance, it will be very beneficial.

DNA Libraries

-A genomic library is a collection of clones that contains every DNA sequence in a genome (cut into fragments). -A complementary DNA (cDNA) library is an entire collection of cloned cDNAs made from the mRNAs isolated from a cell. (So we treat the isolated mRNA with reverse transcriptase that can co from RNA to a DNA copy. Then we use DNA polymerase to fill in the other strand of DNA on all those one-stranded DNA copied from RNA. No we have a collection of ONLY those genes that are actually being expressed in one particular cell at one time). *can be used to tell what genes are expressed at different times of day, in different types of cells, in sick vs. healthy people. -A cDNA library includes only the genes that were active in the cells used as the starting point for creation of the cDNA.

Gel electrophoresis

-a technique that separates DNA, RNA, and protein in a gel with an electrical field (a positive electrode at one end and a negative electrode at the other. *Agarose gel is made into trays with little wells for samples. DNA, RNA, and protein can move through the gel. DNA is negatively charged b/c the links in the backbones are negative, so it will move faster towards the positive electrode, and smaller pieces will move faster than larger. so you get a staggering across the gel plate of molecules sorted by size and polarity.

Molecular Testing Continued: Polymerase Chain Reaction

-shorter faster way to test for genetic disease The polymerase chain reaction (PCR) produces an extremely large number of copies of a specific DNA sequence without having to clone the sequence in a host organism. PCR is essentially DNA replication in which a DNA polymerase replicates only a portion of a DNA molecule. DNA polymerase can't start a new chain, it can only elongate from a preexisting 3' end. So you can make 2 primers, one for each strand, to give the polymerase an easy starting point over the target DNA sequence. The primers used in PCR are designed to isolate the sequence of interest: by cycling 20 to 30 times through a series of steps, PCR amplifies the target sequence, producing millions of copies

Genetically altering animals

-you can introduce a new gene into somatic cells (body cells) so that if the animal has offspring they will not inherit the new gene -OR you could put it into germ-line cells (sperm and eggs) so that the offspring has it in all of their cells, somatic and germ-line. then it can be manipulated through generations with traditional mendelian crossing.

What does an entering plasmid vector need to be replicated by a bacterium?

1) Needs an origin of replication, a place in the DNA for polymerase to latch on and start (same as with the chromosome) - can come in low or high copy number per cell (a plasmid with 200 starting points for replication will reproduce itself way faster than if it had 15) 2) Plasmids are not replicated stably, particularly at low copy numbers. they don't have a mechanism to ensure that some copies go into each daughter during cell replication, so you could get all in one or unequal numbers in the two daughters. 3) like with parasites, replicating that extra plasmid DNA takes time and energy. the cells with only their own DNA and no plasmids will do better. any cell that loses the plasmid will replicate more rapidly and take over environment EXCEPT if there is some survival benefit to having the plasmid, making it worth the extra energy.

Three easy methods to introduce new DNA into a plant:

1) tissue slide: the method discussed earlier 2) flower dip: while plant is flowering (producing seeds) you dip in a medium with agrobacterium with desired gene, and 1-2% of the new seeds will be transgenic 3)biolistics: you introduce new DNA directly into nucleus by shooting tungsten particles coated in the desired DNA into the nucleus and through various mechanisms it can recombine into the chromosome.

Steps of PRC:

1)melt DNA into 2 single strands 2)add a short primer that will bind right next to the target sequence. make two that are compliments of each other, so it will happen on both strands 3) The DNA polymerase will bind to the primer and start replicating and go to the end of the strand. SO the two sides are not matching, they overlap and hang off either end b/c replication started not at the beginning of the strand but at the added primer 4)Now we have two double strand DNA's (but the new halves are not identical to the old halves) 5) melt again, so you now have 4 single strands 6) add more primers 7)DNA polymerase replicates again 8) now we have 4 double strand molecules, and when we melt again we will have 8 templates 9) Now we have 8 double stranded DNA, only two of which are full and identical to the original. Most are modified to amplify the area between the two primers. ***After 3 cycles, the promotion of DNA that is the target area starts increasing exponentially. You can run this cycle 30 times until you have waaaay more of one section of DNA than any other section (the section between the two primers)

Agrobacterium-mediated transformation

Agrobacterium is a bacterium that infects wound sites on trees and causes a sickness by transferring some of its DNA into the plant. It forces the plant cells to grow a tumor that the bacteria then uses as food. It uses Ti plasmid (tumor inducing plasmid) to introduce genes to the plant. Humans can introduce YFG (your favorite gene) into a plant instead of the tumor inducing gene, along with the antibacterial marker that is always needed. So it is a very easy way to make transgenic plants. Through recombinant DNA techniques we can introduce virtually any gene or combination of genes into Agrobacterium and then into many (but not all) plant species.

But sometimes we want a eukaryotic host to grow the desired protein

Could use yeast, or cultured mammalian cells (from mammals). -Hamster mammalian cells are typically used, when sugars/carbohydrates need to be attached to decorate proteins to humanize them.

Producing animal clones

Dolly, the cloned sheep. They took an egg from one, pulled out the nucleus, and introduced a new nucleus from another sheep's adult somatic cell, implant that into an early pregnancy sheep, and dolly was born with the exact genome of the animal from which the nucleus came (as opposed to being born with half the mother's genome, half the father's genome). A clone! BUT there are a lot of mutations that come with the transference of the nucleus. Clones are plagued with health problems cause by flaws in transfer of genome. still, it is entirely possible to clone animals.

Engineering bacteria to produce proteins that we want to use: E. coli example

Engineering E. coli to make a foreign protein: 1) A cDNA copy of a eukaryotic gene for the desired protein is cloned from the appropriate organism. 2) The gene is inserted into an expression vector (plasmid) which contains regulatory sequences that allow transcription and translation of the gene in a host cell. 3) The recombinant plasmid is combined with E. coli, and the E. coli undergoes transformation (takes on the new genes). 4) cDNA is expressed in E. coli, transcribed, and translated to make the encoded eukaryotic protein. That E. coli can now be grown into many colonies that all produce desired protein. 5) The protein is extracted from bacterial cells and purified, or purified from the culture medium ****E. coli is very desirable as a producer of cloned proteins: Can produce eukaryotic proteins that do not require post-translational modification. Is very good at producing well and fast. Is used to make human insulin.

Examples: fireflies

Fireflies make an enzyme called luciferase that allows it to glow in the dark. you can put that glow in the dark gene into plants! it is not that bright though.

Genetic Engineering

Genetic engineering involves manipulating genes for practical purposes. Types: 1)Gene cloning leads to the production of multiple identical copies of a gene-carrying piece of DNA. 2)Recombinant DNA is formed by joining DNA sequences from two different sources. -So we need some molecular "scissors and glue" that will cut up the bits of DNA we want to use and glue them somewhere else.

Review of Genetic Engineering

Genetic engineering uses DNA technologies to modify genes of a cell or organism - organisms that receive genes from an external source (transgenes) are called transgenic. Genetic engineering used to produce proteins used in medicine and research; to correct hereditary disorders; and to alter animals and crop plants.

Genetically modified organisms.

Genetically modified organisms (GMOs) contain one or more genes introduced by artificial means. Transgenic organisms contain at least one gene from another species. GM plants could have: Resistance to herbicides; Resistance to pests; Improved nutritional profile. GM animals could have: Improved qualities; Production of proteins or therapeutic organic materials used in medicine. Reasons why we might need them: we are harming our agricultural land, losing agricultural land area, and increasing our population. food crisis?

Plasmids

Plasmid: small circular DNA molecules that are replicated by bacteria independent of chromosome replication. *discovery: problem of drug resistant bacteria in hospitals making people sick inside the hospital. they proliferate frighteningly fast. study revealed that the antibiotic resistant mutation was not on the bacterial chromosome but on plasmids. resistance was spreading so fast because resistant plasmids were not only spreading through reproduction (cell division), but were being transferred around laterally between bacteria.

Restriction Endonucleases mechanisms

Recognition sequences are the site where the endonuclease cuts the DNA. They are almost always palindromes. EcoR1= 5'-GAATTC-3' Sma1= 5'-CCCGGG-3' .............3'-CTTAAG-5' ...........3'-GGGCCC-5' *likelihood of this formation: 4x4x4x4x4x4=1/4096 will be an EcoR1 recognition site, so they can cut relatively frequently. ***EcoR1 and Sma1 will only cut their recognition sequence if it HASN't been methylated by a modification enzyme, and so must be from outside. EcoR1: Cuts top strand between G and A, bottom strand between A and G, so there are two 5' overhangs, top is AATT, bottom is read the other way so also AATT Sma1: cuts straight through so no overhang. The overhangs are "sticky", ready to bond again to matching sequences (other places where EcoR1 has cut) so a short section that has been cut on both ends can fit into a circular genome that has been cut in one place, be knitted together into it by a ligase enzyme, and now you have a recombinant DNA molecule.

Recombinant DNA

Recombinant DNA can be made by combining DNA from 2 different organisms, or different locations on the same genome. One source contains the gene that will be cloned. Another source is a gene carrier, called a vector. Common vectors of DNA: -Plasmids (small, circular DNA molecules independent of the bacterial chromosome). -bacteriophages

Restriction Endonucleases

Restriction endonucleases are the method of cutting and glueing that is used to recombine DNA into a new pattern in plasmids (the first step of cloning) Derived from system of bacterial defense against bacteriophage viruses (Self vs. non-self: A bacteriophage injects DNA from outside into cell. So you need a mechanism to tell if your DNA is yours or from outside). Two processes form the "restriction modification system" that detect outside DNA and protect cell from it: 1)A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA at specific recognition base pair sequences known as restriction sites. 2)Host DNA is modified by methylation of some of the nucleotides in the restriction site (cutting site) by a modification enzyme (a DNA methyl's), which prevents the enzyme from cutting at the site and so protects host DNA from the restriction enzyme's activity. *So host DNA is methylated to protect it from being cut at the restriction site by endonuclease. Invading bacteriophage DNA is unmethylated and gets digested (cut up), but host DNA is protected.

Molecular Testing for Human Genetic Diseases: Southern Blot Testing

Southern Blot Testing The sickle-cell mutation changes a restriction site in the DNA - Cutting the β-globin gene produces two DNA fragments of the normal gene and one fragment of the mutated gene (more smaller cut pieces come from the healthy gene). So if you can clone a hemoglobin gene and make a probe, you can purify DNA from one homozygous healthy, one homozygous sickle cell, and one heterozygous, use gel electrophoresis to resolve each by size separately to make 3 distributions on gel that you can compare. *The homozygous sickle cell has fewer and longer pieces of cut DNA, so there is one band, farther from positive electrode. *The homozygous healthy has two bands farther from electrode, b/c it is cut into two smaller pieces of slightly different sizes *The heterozygote has all 3 bands, because all 3 sizes are present. **Now you can do this for individuals and compare the patterns to see if they have 0, 1, or 2 sickle cell genes. ***This same test but done with RNA instead of DNA is called northern blot testing

Reproducing Recombinant Plasmids to complete cloning

Steps of cloning a gene: Restriction Modification System: 1)Plasmid DNA is isolated 2)DNA containing the gene of interest is isolated 3)Plasmid DNA is treated with restriction enzyme that cuts in one place, opening the circle 4)DNA with the target gene is treated with the same enzyme and many fragments are produced (not all of which contain the gene of interest) 5) Plasmid and target DNA are mixed and combined with each other 6) Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together (only some of the recombined plasmids contain the gene of interest) Transformation: 7) The recombinant DNA is taken up by a bacterial cell 8)The bacterial cell reproduces to form a clone of cells

Problem with method

With original method, you needed to heat and cool it at EVERY step! really slow and annoying. If you could make an enzyme that doesn't denature at high temperature, you cold just let a machine do it for a few hours without worrying. They found such an enzyme living at really hot temperatures in geysers. Now, it still takes a few hours, but can be run without fail by a machine.

Using PCR to identify an individual's DNA

YOu can obtain a "DNA fingerprint" using STR loci (a Short Tandem Repeat locus is a sequence of repeated nucleotides), to identify an individual. You can use PCR to greatly amplify one tiny area (a locus) that is typically different in different people, so you amplify the differences. In the US, 13 specific loci are used in DNA fingerprinting, and if all 13 match it is very likely that it is DNA from the same person. ***The loci used are 13 different STR loci (short tandem repeat sequences), like CACACACA or GTAGTAGTA. Each locus has different letters repeated, and people have different numbers of repeats, and may be hetero or homozygous for various alleles of that particular locus. Use primers to greatly amplify only these 13 areas, so you have a sample that is almost entirely unique to one person. Use gel electrophoresis to make a distribution pattern for the sample and compare it to a database to find a match.

Pharm animals

You can clone HUMAN protein genes for protein therapy, rather than use cow, etc. proteins that don't work as well. Farm animals genetically modified to produce human rather than animal proteins in their milk, so they are farmed for pharmaceuticals. Purification is easy and harmless to animals, because only mammary cells are altered and proteins are gathered by milking.

Regulation of gene expression in GMO's: Modularity

You can introduce the desired gene, but you must also introduce regulatory elements that can help it to express. Modularity: you can hook up the regulatory elements of another gene in the host organism to the new gene so that it will be expressed where and when you want it to. *you can attach YSFG (your second-favorite gene) that is very easy to tell when it is expressed to YFG (the desired gene) and have them be activated by the same mechanism, and put both into host. The YSFG is a reporter gene that easily marks whether YFG is being expressed (like a gene for a color-changing property, or a gene that will allow it to live in certain conditions that would kill others (like anti-bacterial resistance). then you can put all offspring in those conditions and you know for sure that the ones that live all have YSFG and YFG expressed). With modularity, you can mix and match genes, promoters, regulators.

How to discern which cells that have recombined DNA actually possess the gene of interest

You take all the colonies that have recombined plasmid DNA, and you put them together on a plate in separate areas, and you add chemicals that melt the plasmids apart so you had single stranded loops of plasmid DNA. YOu immobilize them on a membrane You can make a single-stranded DNA piece of the gene of interest, that will bind to the single stranded DNA that also contains the gene of interest. This is called the hybridization probe. You put one strand of this in a bag filled with medium, with the membrane, and the strand will float around until it finds its complement strand (the one recombined with the gene of interest) and bind to it. Then you detect where the one in a million strand is that contains the desired sequence. ***Now that we have found the cloned plasmid with the desired gene, we can take it and reproduce it as much as we want. People use these new cells to make tons of different types of enzymes that perform different functions.

how to discern which cells successfully took on a recombined plasmid

lac Z makes beta-galactosidase cleaves lactose, but also other chemicals with similar linkage. There is a chemical that is clear but turns blue when cleaved by beta-galactosidase. So if you try to recombine a plasmid w/ the lac Z gene by cutting in the middle of it, in ones that do not take on a new section of DNA but simply reform as they were before the lac Z gene will still work and the colony will turn blue. *Now, you can just look at the clear colonies, which have definitely recombined but maybe not with your gene of interest. Now you just have to identify the gen of interest

Example: pBR322 plasmid

pBR322 plasmid: is circular, has convenient cutting sites and codes for cutting (EcoR1), has origin of replication, has two drug resistance genes: 1) resistance to ampicillin and similar drugs. The plasmid teaches host cell how to make a protein that cuts up the ampicillin molecules. 2) tetracycline and similar drugs resistance. teaches host cell how to make transport proteins that can carry the molecules out of the cell. *For recombining of the plasmid, we need those "molecular glue and scissors", or convenient cloning sites where it can be cut and put back together, called restriction sites, and enzymes to do the cutting, called restriction endonucleases. EcoRI is a restriction enzyme that performs this cutting, coded for on the pBR322 plasmid that it is very easy to cut (6 base-pairs long).

Stem Cells

They are capable of undergoing many many divisions undifferentiated, without specializing, and then specialize into anything. Embryonic stem cells are toti-potent: they can become everything, every type of cell that the embryo needs. Adult stem cells function to replace the specialized cells in different types of tissue. *You can grow embryonic stem cells from ice in culture, introduce new genes to them, then reintroduce the transgenic cells to an embryo in a mother mouse, and she will give birth to some babies with original genome, some babies that are transgenic. The new baby mice are called "chimera", DNA from multiple organisms. So some cells from different parts of one mouse will be regular, others transgenic. Two different genomes in one organism. You study the chimeras and hope that some of the germ cells are transgenic. Those will breed the exact same way any other germ cells would, passing on the transgenic traits. *If you mate a transgenic parent with a regular parent, and the progeny are all heterozygote, half regular and half transgenic genome in every cell of the body. ***Scientists have already engineered mice with different qualities.