Unit 11: Biotechnology

1. Unit 11: Biotechnology

2. Introduction • The DNA unit known as nucleotide consists of a five-carbon sugar (deoxyribose), a phosphate group, and one of 4 nitrogenous bases (adenine, guanine, cytosine, thymine). • DNA is formed of two strands of nucleotides combined in a specific way, A with T and C with G. • The strands run opposite, one goes from 5’ to 3’ while the other goes the opposite way, from 3’ to 5’, making the double helix form known today. • RNA unit consists of a five-carbon sugar, a phosphate group and one of 4 nitrogenous groups as well, the difference with DNA is: • The sugar is ribose instead of deoxyribose • In its bases instead of thymine uses uracil • It is not a double strand but a single strand • There are 3 types: messenger , ribosomal and transfer RNA

3. DNA replication • Known as semi-conservative Parent DNA replication Daughter DNA Each DNA strand of the parent cell is used as a template for the 2 daughter cells’ DNA DNA replication occurs inside the nucleus of the cell Daughter strands contain one “old” and one “new” strand.

4. Transcription • Is the process by which the information in the DNA is copied into a strand of messenger RNA (mRNA). • What would have been thymine is changed to uracil in RNA. • Occurs inside the nucleus of the cell. • Example: 5’ TTTAAACCCGGG 3’ DNA 3’ AAAUUUGGGCCC 5’ mRNA

5. Translation • Is the change from a chain of nucleotides to that of amino acids, forming proteins. • Occurs in the ribosomes. • Ribosomes are made of protein and rRNA, and have 2 subunits. • The mRNA passes between the subunits and it is “read” by the ribosome. A codon is made of 3 bases. • tRNA has anti-codons that complement the codons and it carries the amino acid correspondent to it. • As the mRNA is read, the amino acids attach to each other forming a chain which will become a protein.

6. 1- tRNA carries the amino acid to the ribosome • 2- The anticodon of the tRNA is complementary to the codon of the mRNA • 3- As the tRNA moves along the ribosome it releases the amino acid already attached to the prior one. 3 1 2 biologycomer.com

7. Isolation of DNA from banana cells • In a blender the banana is combined with 250 ml deionized water and blended for a few seconds. • 25 ml of a soap solution and 25 ml of a meat tenderizer solution are then added to the banana mixture and mixed gently. • The solution is then heated until it simmers, and filtered. Meat tenderizer and soap are used in the breaking of the cell and nuclear membranes. It is heated to accelerate the process.

8. 10 mL of the filtered solution is put in a test tube • Ice cold 95% ethanol is added to the tube, gently. DNA is not soluble in alcohol, this causes it to come out of solution. Ethanol DNA Filtrate layer

9. Electrophoresis • DNA fragments can be cut by enzymes and then separated by a procedure called gel electrophoresis. Restriction endonucleases or restriction enzymes (REs) are enzymes obtained from bacteria that physically cut DNA. • RE’s recognize a 4- or 6-base pair sequence known as palindromeand cut the DNA in the same way every time. • Some cut through the complementary strands at the same position, producing blunt ends. Others cut it unevenly producing sticky ends.

10. Blunt ends Haemophilus aegyptius (bacteria) produces a restriction enzyme, Hae III that recognizes the palindrome …GGCC… and cuts between the G and the C producing blunt (or flush) ends. …G G C C… …GG + CC… …C C G G… …CC GG… • Sticky ends Escherichia coli RY13 (bacteria) produces a restriction enzyme, EcoR I that recognizes the palindrome … GAATTC… and cuts between the G and the A producing sticky (or staggered) ends. …G A A T T C… …G + AATTC… …C T T A A G… …GTTAA G…

11. Gel electrophoresis • Electrophoresis is the process of applying voltage to a solution of charged molecules (DNA or proteins). • Fragments of DNA obtained by the use of REs are used to identify individuals. • Agarose is used to make a gel where wells are made and the DNA is placed in them. • An electric field is applied and DNA moves to the positive electrode, since DNA is negatively charged. wells Agarose gel

12. Set up Electrodes are used to create an electric charge. DNA will move towards the positive one. Source of electric current + Agarose with wells filled with DNA for testing (outlined by the pink rectangle) is submerged in a conductor of electricity for at least one hour.

13. The DNA fragments move at different rates depending on their size. • Small fragments migrate faster than larger ones. Fragments of the same size concentrate in one group forming a band (thin line) on the gel. well faster moving fragments of DNA + Slower moving DNA fragments DNA movement

14. Reading the results • Population: DNA of the population of the area • Victim: female victim of rape • S/V: DNA of victim and suspected rapist. • S1: suspect 1 • S2: suspect 2 • Which of the 2 you think is the rapist? Population Victim S/V S1 S2

15. Answer • The red rectangle shows the DNA of the victim is present in the S/V sample. • The black rectangles shows the DNA of the rapist coincides with that of suspect 1, concluding he is the rapist. Population Victim S/V S1 S2

16. Importance of gel electrophoresis • Examples where this technique is used in: • Parental disputes • An African-American man in his will asked to be buried in the President Jefferson’s family plot alleging to be a descendant of his, and was denied by the Jefferson’s family. DNA testing proved he was descendent of Thomas Jefferson and Sally Heming so the Jefferson family had to allow it. • Crime investigation • Rapes, incest, murder, etc. • Studies on evolution (how similar or different species are) • Did you know 50% of your DNA is common with that of plants? • Did you know we have at least 20 genes in common with bacteria? • Studies on inheritable diseases • cystic fibrosis, Tay-Sacs, etc.

17. Blood types • ABO blood type group is an example of multiple allele system where 3 alleles determine the outcome. • Follows the principles of co-dominance, in which the presence of two non identical alleles result in the expression of both. • Red blood cells (RBC) have unique antigens on their surface that identifies them as blood type A, B, AB or O.

18. Genetics • Letter I represents the dominant gene and i is the recessive. The exponential represents the blood type present in it. • Both dominant traits are expressed • IAIA and IAi = group A • IBIB and IBi = group B • IAIB = group AB • ii = group O

19. Rh factor is independent from ABO blood type group • Behaves like a complete dominance • DD, Dd = Rh + • dd = RH – • Example • Possible genotype of blood type A+ • IAIADD • IAIADd • IAiDD • IAiDd

20. Blood types, antigens and possible donors Not just anyone can give you blood. Your RBC have antigens on its surface, unique to your blood type, and antibodies that will attack RBC of other blood types as those have different antigens on their surfaces.

21. Determining blood types • Equipment Anti A Anti B Anti Rh Blood sample Chamber s Reactants for group type and Rh

22. Experimentation • A drop of blood is placed in all of the chambers. • In the chamber named A, coagulant for group A is placed, if it is blood type A it will cause it to coagulate. • In the chamber B, a coagulant for group B is placed, if it is blood type B it will cause it to coagulate. • In the chamber Rh, a coagulant for the Rh factor is added, if the blood is Rh+, it will cause it to coagulate.

23. Results Notice the coloration in the wells, it is not uniform or smooth. That indicates coagulation. Remember: coagulation indicates positive for that group This person is AB+

24. Although the colors may seem different, the texture is the same, no coagulation. This person in O- The End