Important Questions 1 What are the building blocks of DNA and how is DNA formed The building blocks of DNA are 2 deoxyribose phosphate groups phosphoric acid and the four bases A T C G Phosphate group ribose sugar and bases Adenine and Guanine are purines Cytosine and Thymine are pyrimidines 2 DNA is positively or negatively charged and why DNA is negatively charged because of the negative charge on the oxygens within the phosphate groups in the DNA backbone The negative backbone strands of DNA want to repel each other this makes DNA unstable Adding Na into solution which neutralizes the negative charges on the backbone and makes the molecule more stable may stabilize DNA 3 How do we identify which is the 5 and the 3 end of a strand of DNA The 5 end has the free phosphate group and the 3 end has a free hydroxyl OH group 4 What does Chargaff s rule state regarding the purine and pyrimidine bases Chargaff s rule is that the amount of Adenine always equals the amount of Thymine and that the amount of Guanine always equals the amount of Cytosine 5 IF you have 100 base pair strand of DNA and know that it contains 44 adenine bases how many cytosine guanine and thymidine bases does the strand contain It must contain 44 thymidine bases and and 66 cytosine and guanine bases 6 Adenine and Thymidine form 2 hydrogen bonds Guanine and Cytosine form 3 hydrogen bonds Answer Hydrogen bonds 7 Which bond is stronger A T or C G and why A C G bond is stronger because a C G bond contains 3 hydrogen bonds as opposed to only 2 hydrogen bonds 8 Order the following bonds in decreasing bond strength hydrogen covalent Van der waals ionic COVALENT is the strongest hydrogen bonds Ionic bonds Van der Waals interactions weakest Covalent bonds are formed between bases and deoxyribose Hydrogen bonds are formed between bases Van der waals interactions are electron cloud interactions between the planar bases within the inside of the DNA molecule 9 A Barr body is an example of Heterochromatin or Euchromatin A Barr body is an example of Heterochromatin Heterochromatin is highly condensed DNA that is so tightly condensed that gene expression is unable to occur because no proteins can access the bases in the DNA A Barr body is a tightly coiled X chromosome that is rendered nonfunctional in females The Barr body that is chosen is completely arbitrary 10 How do histones interact with DNA Histones interact with DNA in the minor groove of the DNA molecule Histones are proteins that are positively charged and they can thus bind and interact with the negatively charged molecules in the minor groove of DNA They interact with DNA at the minor groove because they have tails that allow them to fit nicely into the minor groove Moreover other proteins such as transcription factors methylases and restriction enzymes take up residence in the MAJOR groove of DNA where they are capable of accessing more genetic information 11 How does a specific restriction enzyme know where to cut DNA A specific restriction enzyme knows where to cut DNA because they have very specific restriction sites that they target within a DNA strand Restriction enzymes cleave DNA at particular sites by recognizing specific sequences After recognizing and cleaving DNA the restriction endonucleases leave the cut DNA molecule with sticky ends which are either a 5 overhang or a 3 overhang These sticky ends have complementary base pairs which would allow them to anneal at a later time or be combined with other complementary DNA molecules If a restriction endonuclease cuts a DNA molecule within a restriction site that is very small it may produce blunt ends 12 If you wanted to perform electrophoresis on a sample of fragments of DNA would you load the DNA in the end or the end of the electric field You load DNA in the negative side DNA is negatively charged The other end of the gel is positively charged Thus when an electric field is applied to the gel the DNA travels toward the positive site of the gel DNA fragments that are larger will travel through the gel more slowly and will thus not travel as far during a given period of time 13 What is the difference between ribose sugar in RNA and DNA The ribose sugar in RNA has a hydroxyl group on the 2 carbon However there is no oxygen present at the 2 carbon in the ribose sugar in DNA Thus this is why the sugar in DNA is known as 2 deoxyribonucleic acid it is due to the fact that there is NO HYDROXYL GROUP OH at the 2 carbon on the sugar molecule 14 What makes uracil unique compared to all the other 4 bases Uracil is a pyrimidine molecule Uracil is essentially thymine except it lacks a methyl group at position 5 the 5 methyl group Uracil is only found in RNA and it binds with adenine Uracil is unique because it can also form non Watson Crick base pairs with Guanine in RNA Uracil can also form a triple base pair with adenine forming the structure U A U this triple base pair contributes to the complex tertiary structure of some RNA molecules Hydrogen bonds are the type of bonding that allows Uracil to bind with guanine 15 What forces contribute to the stability of DNA molecules Of RNA molecules Van der Waals interactions hydrogen bonds ionic bonds and covalent bonds all contribute to the stability of DNA Hydrophobic interactions between base pairs in DNA also contribute to the stability of DNA molecules RNA molecules are stabilized by hydrogen bonds covalent bonds ionic interactions and coaxial stacking of the bases RNA molecules are also stabilized when they fold over on each other and form complementary bonds between bases Helical structure and coaxial stacking contribute to the stability of RNA molecules 16 What is the purpose of histone proteins Histone proteins help neutralize the negative charges on DNA they also act as a organizational structure to organize DNA and get it all compact so that it can be wound up tightly within the cell Histone proteins bind with a histone linker protein and 2 DNA strands in order to collectively form a nucleosome Histones package and order DNA up into tightly packed structural units known as nucleosomes 17 Recombinant Plasmids ORI origin of replication Selectable Markers selectable markers can be put into plasmids so that once bacterial cells are transformed with those plasmids it can be determined which cells took up the recombinant plasmids and which ones did not Selectable markers are often sequences that confer antibiotic resistance or fluorescence Insertion Sites this is where