DNA Scisssors Introduction to Restriction Enzymes

NAME:_____________________ DNA Scissors: Introduction to Restriction Enzymes Genetic engineering is possible because of special enzymes that cut DNA. These enzymes are called restriction enzymes. Restriction enzymes are special proteins produced by bacteria to prevent or restrict invasion by foreign DNA (such as from viruses). They act as DNA scissors, cutting the foreign DNA into pieces so that it cannot function. Restriction enzymes recognize and cut at specific places along the DNA molecule called restriction sites. Each different restriction enzyme (and there are hundreds, made by many different bacteria) has its own type of restriction site. In general, a restriction site is a “4” or “6” base pair sequence that is a palindrome. A DNA palindrome is a sequence in which the “top” strand read from the 5’ end of DNA to the 3’ end is the same as the “bottom” strand read from the 5’ end of DNA to the 3’ end (i.e. it reads the same forward and backward). For example: 5’ GAATTC 3’ 3’ CTTAAG 5’ is a DNA palindrome. To verify this, read the sequence of the top strand and the bottom strand from the 5’ end to the 3’ end. This sequence is also a restriction site for the restriction enzyme called EcoRI. Its name comes from the bacterium in which it was discovered. Eschcerichia coli strain RY 13 (EcoR), and “I” because it was the first restriction enzyme found in this organism. Check for Understanding I: 1. What type of molecule is an enzyme? ____________________________ 2. What kind of enzymes make genetic engineering possible? ________________________ 3. What is the function of these enzymes? ___________________________________ 4. What is a restriction site? _____________________________________________ 5. What would be the opposite strand of the following DNA palindrome? 5’ CAATTG 3’ 3’_________5’ The restriction enzyme EcoRI makes one cut between the G and A in each of the DNA strands (see below). After the cuts are made, the DNA is held together only by the hydrogen bonds between the four bases in the middle. Hydrogen bonds are weak, and the DNA comes apart. cut sites: 5’ GAATTC 3’ 3’ CTTAAG 5’ cut DNA: 5’ G AATTC 3’ 3’ CTTAA G 5” The EcoRI cut sites are not directly across from each other on the DNA molecule. When EcoRI cuts a DNA molecule, it therefore leaves single-stranded “tails” on the new ends (see the example above). This type of end has been called a sticky end because it is easy to rejoin it to complementary sticky ends. Not all restriction enzymes make sticky ends; some cut the two strands of DNA directly across from one another, producing a blunt end. When scientists study a DNA molecule, one of the first things they do is to figure out where many restriction sites are. They then create a restriction map, showing the location of cleavage sites for many different enzymes. These maps are used like road maps to the DNA molecule. Following are the restriction sites of several different restriction enzymes, with the cut sites shown: EcoRI: 5’ GAATTC 3’ 3’ CTTAAG 5’ HindIII: 5’ AAGCTT 3’ 3’ TTCGAA 5’ BamHI: 5’ GGATCC 3’ 3’ CCTAGG 5’ AluI: 5’ AGCT 3’ 3’ TCGA 5’ SmaI: 5’ CCCGGG 3’ 3’ GGGCCC 5’ HhaI: 5’ GCGC 3’ 3’ CGCG 5’ Knowledge of these various restriction enzymes has enabled genetic researchers to create recombinant DNA which is DNA made by recombining fragments of DNA from different sources. Industry, medicine, and agriculture have all been enhanced with the creation of recombinant bacteria (living bacteria that contain recombinant DNA and thus can make useful proteins). In industry, recombinant bacteria have been engineered that will break down oil and help clean up oil spills. Mining companies are trying to use this technology to engineer bacteria that will extract valuable minerals from ores. In medicine, recombinant DNA is being used to produce various hormones, such as human growth hormone and insulin. The gene for human insulin is inserted into a bacterium and as these bacteria live and reproduce normally, they produce large quantities of insulin that is used by diabetics. In agriculture, recombinant bacteria are helping to prevent frost damage in some crops and helping to convert atmospheric nitrogen to nitrates, a natural fertilizer used by plants. Check for Understanding II: 6. What is a “sticky end”? ______________________________ 7. Which of the enzymes above would leave sticky ends? ________________________ 8. Which of the enzymes above would leave blunt ends? _________________________ 9. What is recombinant DNA? _____________________________________________ 10. What is one use of recombinant DNA? _____________________________________ 11. Would DNA with sticky ends or blunt ends be used to make recombinant DNA? __________ Explain why: Paper Simulation of Restriction Enzyme Action PROCEDURE PART I: 1. Cut out the DNA strips along the borders. These strips represent double-stranded DNA molecules. 2. You will now simulate the activity of EcoRI. Scan along the DNA sequence of strip 1 until you find the EcoRI site (refer to the list above for the correct sequence). Make cuts through the sequence of bases by cutting just between the G and first A of the restriction site on BOTH STRANDS. –DO NOT CUT ALL THE WAY THROUGHT THE STRIP! Remember that EcoRI cuts the backbone of each DNA strand separately. 3.Now separate the hydrogen bonds between the cut sites BY CUTTING THROUGH THE VERTICAL LINES. Separate the two pieces of DNA. Look at the new DNA ends produced by EcoRI. 4.Write “EcoRI” on the cut ends. Keep these fragments on your desk to use later. 5.Repeat the procedure with strip 2, this time simulating the activity of SmaI. Find the SmaI site and cut through the bases at the cut sites indicated above. Label the new ends “SmaI” and keep the DNA fragments on your desk. 6.Simulate the activity of HindIII with strip 3. Label the new ends “HindIII” and keep the fragments. 7.Repeat the procedure once more with strip 4, again simulating EcoRI. QUESTIONS PART I: 1. Are the EcoRI ends sticky or blunt?__________________________ 2. Are there any hydrogen bonds between the cut sites in SmaI? ______________________ 3. Are the SmaI ends sticky or blunt? _______________________ 4. Are the HindIII ends sticky or blunt? __________________________ 5. Pick up the “front end” DNA fragment from strip 4 (an EcoRI fragment) and the “back end” HindIII fragment from strip 3. Both fragments have single-stranded tails of 4 bases. a. Write down the base sequence of the two tails and label them “EcoRI” and “HindIII’, label the 5’ and 3’ ends. _________________________________________ _________________________________________ b. Are the base sequences of the HindIII and EcoRI “tails” complementary? ___________ Explain why this is so: 6. Now pick up the “back end” DNA fragment from strip 1 (strip1 cut with EcoRI), and put down the HindIII strip. Compare the single-stranded tails of the EcoRI fragment from strip 1 and the EcoRI fragment from strip 4. Write down the base sequences of the single-stranded tails and label the 3’ and 5’ ends. ____________________________________________ ____________________________________________ Are these ends complementary?_____________Explain why this is so: 7. Imagine that you cut a completely unknown DNA fragment with EcoRI. Do you think that the single-stranded tails of these fragments would be complementary to the single-stranded tails of the fragments from strip 1 and strip 4. __________________ Explain why you think this to be: 8. There is an enzyme called DNA ligase that re-forms the bonds between the nucleotides. For DNA ligase to work, two nucleotides must come close together in the proper orientation for a bond (the 5’ side of one must be next to the 3’ side of the other). Do you think it would be easier for DNA ligase to reconnect two fragments cut by EcoRI or one fragment cut by EcoRI with one cut by HindIII? _____________________________________ Explain why: 9. Paste one Recombinant DNA molecule below. This recombinant DNA molecule should be connected by sticky ends. Then recycle the other fragments. DNA Sequence Strips for DNA Scissors Color Red Color Green Color Orange Color Brown