U8T5 – Recombinant DNA Technology

Sometimes when trying to find a gene which important in a particular disease, it is useful to find out what genes the diseased cells have used (expressed) by extracting mRNA rather than DNA. However, mRNA cannot be used in genetic modifications. What do we need to change it into to be able to use it?

Which enzyme do we use to convert mRNA into cDNA?

How would the cDNA base sequence of a gene differ to the normal genomic base sequence of a gene?

What do we call enzymes that cut DNA at specific base sequences?

What is the active site of a restriction endonuclease complementary too?

Which bond to restriction endonucleases ( restriction enzymes) break?

What do we call it if the restriction enzyme cuts both strands of DNA in the same place?

What do we call it if the restriction enzyme cuts both strands of DNA in a staggered position to create an overhang?

If the same restriction enzyme is used to make two fragments, what can form between the exposed bases on each end if they are mixed with each other?

Which enzyme can join two complementary sticky ends together during genetic modification?

What bond does DNA ligase create?

Usually the cut out gene is inserted into a vector. Which of the following is a vector for use in genetic modification of bacteria?

If we cut out a human gene using restriction enzymes and ligate it into a plasmid using ligase, then that plasmid is now 'Recombinant'. What do we call the process of putting the recombinant plasmid into bacteria?

Using bacteria is an example of what type of DNA Amplification?

If a bacterium contains a recombinant plasmid, then it will express the new 'foreign' gene as well as it's own. Which of the following is not a product made by recombinant bacteria?

Apart from the desired gene sequence, what else needs to be added to a recombinant plasmid in order for it to be expressed by bacteria?

We cannot see the proteins made by recombinant plasmids, nor the plasmids themselves. How do we know the bacteria have been transformed properly and contain a recombinant plasmid ( = plasmid and new gene fragment) ?

Sometimes, two marker genes are used: The first marker gene is often an antibiotic resistance gene to show if the plasmid is present; the bacteria can only grow on a plate containing the antibiotic, if they have the antibiotic resistance gene on the plasmid. The second is often a gene with a colour change, which is broken by the insertion of the fragment.

e.g. An example of a gene to test for the presence of a fragment is Green Fluorescent Protein (GFP) glowing or not. What would a bacterial plate look like that contained an antibiotic, and was spread with bacteria which had been transformed with a plasmid and had a new gene fragment in?

PCR is an example of what type of DNA amplification?

What enzyme is needed in a PCR reaction ?

In a PCR reaction, the DNA is heated at the start of the cycle to around 95 degrees C. What is the reason for this?

Primers are short single stranded pieces of complementary sequenced DNA. Which ISN'T a reason to add specific primers added to the PCR reaction?

Why do we need two primers in PCR?

PCR allows DNA to replicate by semi-conservative replication. But what word best describes the rate at which it amplifies?

If you start with a single copy of template DNA, how many copies would you have after 10 cycles?