1. Endonucleases usually act on palindromic DNA sequences.

1. What is meant by palindromic sequence?
2. Construct two palindromic sequence?

      1. DNA that have same nucleotide sequence on the two strands in 5' 3' direction; and 3'  5'  direction.

      2.  a. 5'  - GAATTC - 3'

           3' - CTTAAG - 5'

      b. 5' - GGATCC - 3'

           3' - CCTAGG - 5'

2. Identify the following.

1. Molecular glue 
2. Molecular scissors

      1. DNA ligase 

      2. Restriction enzymes.

3. Raju, during his biotechnological experiments, separated DNA fragments through gel electrophoresis.   But he failed to observe the fragment clearly.  As a biology student, suggest a staining procedure for visualizing the DNA fragments. 

     Stain the gel with ethidium bromide.  Then the gel should be exposed under UV light.  The DNA Fragments are visible as orange bands.  

4. During DNA technology experiments, a common restriction enzyme is used to cut both vector and DNA of interest. 

• Do you agree with the statement?
• Justify your answer.

• yes,
• Because  the same restriction enzyme cut specific sequences and create sticky ends in DNA fragments which is useful to ligate the DNA and production of recombinants are easy. 

5. A cloning vector usually contains a particular DNA sequence called Ori.

      a. Mention two characteristics of Ori.

      b. List out other features of a plasmid that makes it a suitable vector. 

       a.  1. It is the region where replication starts.

            2. Ori determines the copy number of recombinants.

        b. Plasmid vector contain cloning sites, antibiotic resistant gene as selectable markers and recognition sites.

6. DNA fragments can be separated by Gel electrophoresis. Explain the process.

DNA fragments are negatively charged molecules and they can be separated by moving them towards the anode under an electric field through agarose gel medium.  The smaller DNA fragments  further move.  These fragments are then stained with ethudium bromide followed by exposure to UV rays, and the orange bands of DNA are clear.

7. Different steps involved in recombinant DNA technology using restriction enzyme are listed below in a jumbled order.  Rearrange them in correct order.

a. Formation of sticky ends.

b. Recognition and cutting of vector and foreign DNA.

c. Formation of recombinant DNA.

d. Ligation of vector and foreign DNA using ligase enzymes. 

b, a, d, and c

8. 'Without restriction enzyme and ligase, recombinant DNA technology is impossible'

a. Do you agree with this statement?

b. Give reason.

a. Yes.

b. Because these are the key enzymes use to cut (restriction enzyme) and join (ligase) DNA fragments which is the basic process in recombination DNA technology.

9. Normally bacterial cell will not take up plasmids, so they have to make it competent for this.

a. Explain the method to make them competent.

b. Name two methods used to introduce alian DNA directly into host cells. 

a. Bacterial cell is treated with a specific concentration of divalent cation (Ca), which increases the efficiency with which DNA enters the bacterium through pores in the cell wall.  Recombinant DNA is then forced into it by incubating the cells with rDNA on ice, followed by placing them at 42^o C and then putting them back on the ice.  Thus, the bacteria take up the rDNA.

b. Micro injection and gene gun.

10. PCR is an important technique in gene cloning.  Write down the purpose of PCR by pointing out the major steps involved in PCR.

     Polymerase chain Reaction (PCR) is used to amplify the desired  DNA.  The major steps in PCR are Denaturation, Annealing and Extension. 

1. Denaturation: The double stranded DNA is separated into single stranded by applying heat.  Each strand act as a template.

2. Annealing: Primers ( Chemically Synthesised nucleotides that are complimentary to DNA strand) are annealed to DNA strands.

3. Extension:  Primer is extended to the length of DNA using polymerase enzyme ( Taq Polymerase).