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 Recombination Technology

 Recombination Technology ¡¡   What is Recombinant DNA? Any DNA molecule formed by joining DNA segments from different sources. What are Plasmids? Small, circular, extrachromosomal DNA molecules. They can replicate independently of the genome, and are found in numbers ranging from one per cell to hundreds per cell (this is called "copy number"). Plasmids frequently carry genes for antibiotic resistance. While antibiotic resistance is becoming an increasingly important problem medically, it is a useful marker in Recombinant DNA technology. Such markers, along with the small size and potentially high copy number, make plasmids indispensible tools in Molecular Biology. The following figure is an example of a typical plasmid, called pACYC184, which is 4,240 base pairs (bp, or 4.24 kilobases, kb). This plasmid map has 3 marked regions on it. Two of them are antibiotic resistance genes, one for Tetracycline resistance (Tc) and the other for Chloramphenicol resistance (Cm). The arrows denote the direction the genes are transcribed. Remember that transcription can only occur in the 5' --> 3' direction, but plasmids are double-stranded, so the direction needs to be explicitly stated so it is known which strand is used as template for transcription. The other marked region (ori) is the origin of replication, the region where the DNA replication machinery assembles. What are Restriction Enzymes? Also known as Restriction Endonucleases, these enzymes recognize and cut specific sequences in double-stranded DNA. Discovered in bacteria in 1962, Restriction Enzymes are made to protect the bacteria from foreign DNA. Bacteria have a method of marking their own DNA as being "self" (called a Modification System). Any DNA not recognized as self is digested into smaller pieces by the Restriction Enzymes. Restriction Enzymes search for exact sequences of a defined length. Some enzymes recognize sequences 4 bp long (e.g., GTAC), some 6 (e.g., GAATTC), and still others 8 or more. One of the common features of most enzyme recognition sites is that they are palindromes. A palindrome is a sequence which is read the same on both strands in the 5' --> 3' direction. This is the recognition sequence for Eco RI. See how the sequence reads the same from either direction? The arrows are the points at which the enzyme actually causes breaks in the backbone of the DNA. Once the backbone is broken, the hydrogen bonds are not sufficient to hold the strands together, and they separate. This type of cut is called staggered, because it results in fragments with single-stranded ends. The single-stranded ends are said to be sticky because they are able to bind to a complementary single-stranded region. The single-stranded overhang from one fragment can be paired with any other fragment of DNA, including one from a different organism, that has a complementary sequence, by joining the two fragments together with another enzyme called DNA Ligase. Restriction Enzymes can also produce fragments without single-stranded ends. This is called a blunt cut. This example of a blunt cut is from Eco RV, another Restriction Enzyme isolated from E. coli. As opposed to sticky cutters, no complementarity is required, so any two blunt ended fragments can be joined by DNA Ligase. Do you notice anything interesting about the names of the enzymes? The first three letters identify the organism the enzyme was isolated from. The first letter is from the genus, the next two are from the species name. For example, the commonly used enzyme Bam HI comes from the bacterium Bacillus amylophilus, and Sau 3A comes from Staphylococcus aureus. How often does a Restriction Enzyme find the sequence it's looking for? It depends on how many bases it's looking for. It's really just a question of probablility. There are 4 bases in any given strand of nucleic acid, right? That means that the probability of finding one of those, for example Adenine, at a particular position is 25%, or 1/4. If you are looking for an Adenine next to a Guanine, then the probability is 1/4 for each position, or (1/4)x(1/4)=1/16. If you are looking for the string of bases AGC, the probability is (1/4)$3$ , or 1/64. This means that every 64 bases, you would expect to find the string AGC. Many, but by no means all, Restriction Enzymes recognize exact sequences of bases 6 nucleotides long (you will often hear enzymes like this referred to as "6-cutters"). How often would you expect an enzyme like this to find what it's looking for? (1/4)$6$ = 1/4096, or about once every 4,100 base pairs Just like most things in the world of statistics, these are only probabilities. So don't think that you can count exactly 4,096 bases from one cleavage site and expect to find the next. But if you start with a linear piece of chromosomal bacterial DNA that is 5,000 kb long (5 million base pairs) and digest it with a 6-cutter like Eco RI, statistically you would expect to end up with roughly 1221 individual fragments (5,000,000 divided by 4,096), and realistically, that number will be pretty close.

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