1.

If human DNA were essentially a random sequence of 5 x 109 bp with equal proportions of all nucleotides, approximately how many restriction fragments would be expected from cleavage with (a) 4-base cutter, (b) 6-base cutter 2. A fragment of human DNA with an EcoRI site at one end and HindIII site at the other end is labeled with 32P at the EcoRI end. This molecule is now partially digested with either restriction enzyme HhaI or Sau3A, and the fragments are separated on agarose gel as shown in figure below a. Draw a map of this fragment showing the locations of the HhaI recognition sites and the distances of the sites from one end. b. The same fragment labeled with 32P in the same fashion is now digested to completion with HhaI, and the products are separated on an agarose gel. Sketch the expected pattern for the fragments on the gel.

3. A cloned DNA fragment of DNA was sequenced by using the dideoxy method. A part of the autoradiogram of the sequencing gel is represented here:

(a) Deduce the nucleotide sequence of the DNA chain synthesized from the primer. Label the 5 and 3 ends b) Deduce the nucleotide sequence of the DNA chain used as the template strand. Label the 5 and 3ends.

4. How would a DNA sequencing reaction be affected if the ratio of dideoxynucleoside triphoshates (ddNTPs) to deoxynucleosides triphoshates (dNTPs) were increased? What would the consequences be if the ratio were decreased?

5. The restriction nuclease Sau3A recognizes the sequence GATC and cleaves on the 5 side (to the left) of the G. The single stranded ends produced by Sau3A cleavage are identical to those produced by BamHI cleavage, allowing the two types of ends to be joined together by incubation with DNA ligase (a) What fraction of BamHI sites can be cut with Sau3A? What fraction of Sau3A sites can be cut with BamHI? (b) If two BamHI ends are ligated together, the resulting site can be cleaved again by BamHI. The same is true for two Sau3A ends. Suppose you ligate a Sau3A end to BamHI end. Can the hybrid site be cut with Sau3A? Can it be cut with BamHI? (c) What do you suppose is the average size of DNA fragments produced by digestion of chromosomal DNA with Sau3A? Whats the average size with BamHI? 6. If cell is known to carry five genes that code for enzymes of the same metabolic pathway. How can one find out if all the five genes are located next to one another on the same chromosome? 7. The following pattern is obtained from DNase footprinting experiment in which either RNA polymerase + repressor or repressor protein alone was added to DNA fragment (containing repressor gene, promoter, operator and structural genes of lac operon), and then the complex was digested with DNase.

8. You wish to know whether the cDNA you have isolated and sequenced is the product of a unique gene or is made by a gene that is member of a family or related genes. To address this question, you digest cell with DNA with a RE nuclease that cleaves the genomic DNA BUT not the cDNA, separate the fragments by gel electrophoresis, and visualize bands using radioactive cDNA as a probe. The Southern blot shows two bands, one of which hybridizes more strongly to the probe than the other. You interpret the stronger hybridizing band as gene that encodes your cDNA and the weaker band as a related gene. When you explain your result to your advisor, she cautions that you have not proven that there are two genes. She suggests that you repeat the Southern blot in duplicate, probing one with the radioactive segment from the 5 end of the cDNA and other with a radioactive segment from the 3 end of the cDNA.

a) How might you get two hybridizing bands if the cDNA was the product of a unique gene? b) What results would you expect from the experiment your advisor proposed if there were a single unique gene? If there were two related genes? 9. You wish to make a restriction map of a 3.0 Kb BamHI restriction fragment. You digest three samples of the fragment with EcoRI, HapII, and a mixture of EcoRI and HapII. You then separate the fragments by gel electrophoresis and visualize the DNA bands by staining with ethidium bromide (fig). From these results, prepare a restriction map that shows the relative position of the EcoRI and HapII recognition sites and the distances in kilobases between them.

10. Look carefully at the structures of the molecules in figure below.

A) What would you expect to happen if dideoxycytidine triphosphate (ddCTP) were added to a DNA replication reaction in large excess over the concentration of deoxycytidine triphosphate (dCTP)? Would it be incorporated into the DNA? If it were, what would happen after that? Give your reasoning. B) What would happen if ddCTP were added at 10% of the concentration of dCTP? C) What effects would you expect if dideoxycytidine monophosphate (ddCMP) were added to a DNA replication reaction in large excess, or at 10% of the concentration of dCTP? 11. A 11 kb DNA fragment cut with HindIII generates fragments of 7.0 and 4.0 kb respectively. The restriction nuclease HindIII cuts the recognition sequences as shown below. You incubate the cut molecule with DNA polymerase and all four dNTPs and then ligate the molecule. Following ligation you digest the molecule with HindIII. What is the size of the DNA fragment obtained upon digestion of the ligated molecule with HindIII? 12. Consider the following statements: A human cell contains 46 molecules of DNA in its nucleus. Do you agree with it? Why or why not?

13. Suppose that you are unable to repair the damage to DNA caused by the loss of purine bases. This defect cvauses the accumulation of about 5000 mutations per day in the DNA of each of your cells. As the average difference in the DNA sequences between humans and chimpanzees is about 1%, it is only a matter of time until you turn into a chimp. What is wrong with this argument? 14. What is so special about RNA that makes it such an attractive evolutionary precursor to DNA and protein? What is so special about DNA that makes it better material than RNA for the storage of genetic information? 15. A molecule of double stranded DNA that is 3 million base pairs long has a base composition that is 48% G +C. How many times on average, is an HpaII restriction site (recognition sequence = CCGG) likely to be present in this DNA molecule?
16. A cloned DNA fragment was sequenced by using the dideoxy method. A part of the autoradiogram of the sequencing gel is represented below: Deduce the nucleotide sequence of the DNA chain used as the template strand. Label the 5 and 3 ends.

ddA

ddT

ddC

ddG

17. For the DNA fragment (other strand complementary of it) shown below, decide whether it will be cut by the restriction nucleases EcoRI (5-GAATTC), AluI (5-AGCT), and PstI (5CTGCAG). For those that cut the DNA, how many products will be produced? 5-AAGAATTGCGGAATTCGAGCTTAAGGGCCGCGCCGAAGCTTTAAA-3 18. To prepare a genomic DNA library, it is necessary to fragment the genome so that it can be cloned in a vector. A common method is to use a restriction nuclease. A). how many different DNA fragments would you expect to obtain if you cleaved human genomic DNA with Sau3A (5-GATC)? (Recall that there are 3.2 x 109 base pairs in the haploid human genome). How many would you expect to get with EcoRI (5-GAATTC)? B). Human genomic libraries are often made from fragments obtained by cleaving human DNA with Sau3A in such a way that the DNA is only partially digested; that is, so that not all the Sau3A sites have been cleaved. What is the possible reason for doing this?

19. You want to amplify the DNA between the two stretches of sequence shown in figure 2a. Of the listed primers choose the pair that will allow you to amplify the DNA by PCR. DNA to be amplified 5- GACCTGTGGAAGC_________________________CATACGGGATTGA - 3 3- CTGGACACCTTCG__________________________GTATGCCCTAACT - 5 Primers 5) 5- CATACGGGATTGA - 3 6) 5- GTATGCCCTAACT - 3 7) 5- AGTTAGGGCATAC - 3 8) 5- TCAATCCCGTATG - 3

1) 5- GACCTGTGGAAGC - 3 2) 5- CTGGACACCTTCG - 3 3) 5- CGAAGGTGTCCAG - 3 4) 5- GCTTCCACAGGTC - 3