Question

You are tasked to clone the human actin gene (blue) from a cDNA clone that you obtained from a collaborator into the cloning vector termed pUC119 using the HindIII restriction enzyme. The 4 blue arrows show the HindIII recognition sequence on the Actin cDNA clone. Plac MCS Hindill Sphl sbil Pst BspMI Accl Hincli Sall Xbal BamHI Smal Xmal Acc651 Kpnl Eco53ki Sacl EcoRI lacz pMB1 ori M13 IG PUC119 3162 bps CDNA Clone Amp a. b. Figure 2: a. pUC119 cloning vector; b. cDNA clone obtained from your collaborator showing HindIII restriction sites (arrows) How would you confirm that the cloning vector contains the complete gene fragment. Please explain fully how you would differentiate between bacterial cells containing the cloning vector with (a) no insert (b) partial actin gene frangment (C) complete actin gene fragment.

Answer 1

1. To confirm if the cloning vector contains the complete gene fragment

The human actin gene according to the above corraborator's picture has four HindIII restriction sites and the cloning vector pUC 119 has a single HindIII restriction site. To check if the vector has the gene insert, the cloned vector needs to be subjected to a restriction digestion using HHindIII restriction enzyme. This digestion will release 4 fragments and can be visualised in a DNA electrtophoresis gel against a DNA marker or ladder of which 3 will belong to the insert and the fourth should match with linearised vector.

a) no insert: There will be no fragment released instead the cloned vector pUC119 will be in its linear form and can be visualised in a DNA electrtophoresis gel against a DNA marker or ladder and a negative control of uncut uncloned vector and another control of uncut recombinant vector.

b}partial actin gene fragment: This digestion will release 4 fragments and can be visualised in a DNA electrtophoresis gel against a DNA marker or ladder of which 3 will belong to the insert and the fourth should match with linearised vector. if there are less than 3 fragments apart from the linearised plasmid fragment band then it means that the full gene was not cloned in.

c)complete actin gene fragment: The digestion will release 4 fragments and can be visualised in a DNA electrophoresis gel against a DNA marker or ladder of which 3 will belong to the insert and the fourth should match with linearised vector, the bands corresponding to the fragment sizes should match against the DNA markers and controls of uncut vector, uncut recombinant vector and the gene alone. Also the uncut successfuly cloned vector with the complete gene will be longer and hence the band will appear in a higher position than the vector without insert or with partial insert. also linearised forms of all the cloned vector and other controls can be linearised with another restriction enzyme and can be checked with sizes.