Recombinant DNA Techniques
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Questions and Answers

What are the tools that allow us to manipulate DNA?

Replication and its enzymes

What is the function of DNA polymerase in recombinant DNA technology?

Create new DNA

What is the primary function of ligase in DNA manipulation?

Joining of molecules, pasting DNA fragments together

Plasmids are large circular DNA molecules found in bacteria.

<p>False</p> Signup and view all the answers

What is the primary function of restriction enzymes in recombinant DNA technology?

<p>Cutting DNA at specific sequences</p> Signup and view all the answers

How are bacterial protection enzymes named?

<p>Based on the bacteria they were discovered in</p> Signup and view all the answers

Give an example of a bacterial protection enzyme against bacteriophages.

<p>EcoR1</p> Signup and view all the answers

What is the purpose of bacteria releasing restriction enzymes?

<p>Cutting up viral genetic material to stop infection</p> Signup and view all the answers

How do bacteria protect their own DNA from being cut by restriction enzymes?

<p>Methylation</p> Signup and view all the answers

What happens when a virus infects a bacteria?

<p>Its genetic material is injected into the cell</p> Signup and view all the answers

Between which sequence does EcoR1 cut DNA?

<p>GAATTC</p> Signup and view all the answers

Why do all restriction enzymes cut at the sugar-phosphate backbone of DNA?

<p>Because the bond between the sugar and phosphate is the weakest link in the DNA molecule</p> Signup and view all the answers

What are the two types of ends that restriction enzymes can leave after cutting DNA?

<p>Complementary overhands and blunt ends</p> Signup and view all the answers

What was the significant experiment conducted by Paul Berg in the 1970s?

<p>He created hybrid DNA by joining E. coli DNA and SV40 viral DNA using EcoR1 and ligase</p> Signup and view all the answers

What is the typical size range of plasmids?

<p>1000-4000 base pairs long</p> Signup and view all the answers

Why are plasmids often used as vectors in research?

<p>They serve as carriers of DNA pieces of interest.</p> Signup and view all the answers

How do you get a gene of interest to replicate with a plasmid?

<p>Cut the plasmid and the DNA of interest with the same restriction enzyme and then ligate them together</p> Signup and view all the answers

What is the next step after a gene of interest is inserted into a plasmid?

<p>Transform the recombinant plasmid into a bacterial cell</p> Signup and view all the answers

What is the first step in the transformation process?

<p>Putting the recombinant plasmid inside a cell</p> Signup and view all the answers

What is transformation?

<p>The process of getting the recombinant plasmid inside a cell</p> Signup and view all the answers

What are the two common methods used to get a plasmid into a cell?

<p>Heat shock</p> Signup and view all the answers

How do you confirm that the bacteria has successfully taken up the plasmid?

<p>Using antibiotic selection</p> Signup and view all the answers

Sticky ends on a plasmid can only correctly ligate to the inserted gene.

<p>False</p> Signup and view all the answers

What confirmation is missing after bacteria have successfully taken up the plasmid?

<p>Whether the gene of interest is also present in the plasmid</p> Signup and view all the answers

What is the function of the gene called beta galactosidase found in some plasmids?

<p>It codes for an enzyme that breaks down lactose</p> Signup and view all the answers

The beta galactosidase gene has a restriction site in the middle of it.

<p>True</p> Signup and view all the answers

What happens if the insert is successfully inserted into the beta galactosidase gene?

<p>It interrupts the beta galactosidase gene</p> Signup and view all the answers

What happens if the insert doesn't go into the beta galactosidase gene?

<p>Beta galactosidase is functional and can code for the enzyme</p> Signup and view all the answers

Describe what happens after the transformation process is complete.

<p>The bacteria are grown on Amp plates (selecting for the plasmid) and an indicator is added to see if lactose is being digested (blue color)</p> Signup and view all the answers

What do plain white colonies signify in the transformation experiment?

<p>Beta galactosidase has been interrupted by your gene of interest</p> Signup and view all the answers

Why is agarose used in gel electrophoresis?

<p>It solidifies into a jello-like substance</p> Signup and view all the answers

What is the first step in gel electrophoresis?

<p>Place the gel into a buffer solution and add your DNA sample</p> Signup and view all the answers

What is the second step in gel electrophoresis?

<p>Run an electrical current through the buffer chamber</p> Signup and view all the answers

Why does DNA migrate towards the positive charge in gel electrophoresis?

<p>DNA has a negative charge</p> Signup and view all the answers

What happens to the DNA fragments as they move through the agarose gel?

<p>They wind their way through the pores</p> Signup and view all the answers

How does gel electrophoresis show the separation of DNA fragments?

<p>The smaller fragments (less base pairs) move through these pores much quicker than larger fragments</p> Signup and view all the answers

Why is a staining agent added to the gel after electrophoresis?

<p>So you can see the fragments under UV light</p> Signup and view all the answers

What is the first feature of a good vector?

<p>Size - smaller than bacterial chromosome</p> Signup and view all the answers

What is the second feature of a good vector?

<p>High copy number - high replication rate = 1000's per cell</p> Signup and view all the answers

Study Notes

Recombinant DNA Techniques

  • DNA Manipulation: Learning replication secrets and enzymes allows manipulating and cloning DNA fragments.
  • DNA Polymerase: Creates new DNA.
  • Ligase: Joins molecules using ATP; "pastes" DNA.
  • Plasmids: Small, circular, extra-chromosomal DNA; self-replicating in bacteria.
  • Restriction Enzymes: Cut DNA at specific reverse palindrome sequences. Bacterial defense against bacteriophages.
  • Bacterial Protection Names: Named after bacteria they were discovered in. Example: EcoR1 (E. coli).
  • Bacterial Restriction Enzyme Function: Cutting viral DNA to stop infection.
  • Bacterial DNA Protection: Bacteria methylate their DNA to prevent self-cutting by restriction enzymes.
  • Bacteriophage Infection: Viral genetic material injected into bacteria.
  • EcoR1 Cut Site: Cuts DNA at the sequence GAATTC.
  • DNA Backbone Cuts: All enzymes cut the sugar-phosphate backbone bond.
  • Enzyme Cut End Types: Some enzymes leave "sticky ends", others yield "blunt ends".
  • Paul Berg (1970s): Created hybrid DNA using EcoR1 and ligase combining E. coli and SV40 viral DNA.
  • Plasmid Size: Typically 1000-4000 base pairs (bps).
  • Plasmid Use: Vectors in research for carrying DNA fragments of interest.
  • Cloning Procedure: Extract gene fragment, digest plasmid and fragment with same restriction enzyme, ligate them together.
  • Plasmid in Cell: Plasmid insertion into a cell to clone a gene and make the gene product.
  • Transformation Methods:
    • Electroporation: Shock cells to introduce plasmid.
    • Heat Shock: Briefly heat-treat chilled cells.
  • Identifying Plasmid-Containing Bacteria: Antibiotic selection; plasmids encode antibiotic resistance. Grow on antibiotic plates.
  • Plasmid Sticky Ends: Sticky ends on a plasmid can self-ligate without the inserted gene.
  • Confirmation of Gene Insertion: Transformation confirms plasmid presence, but not necessarily gene of interest.
  • Second Selection: Plasmid with beta-galactosidase gene (lactose-digesting enzyme); gene interruption indicates gene of interest insertion.
  • Selection for Inserts: Grow on antibiotic plates and look for white colonies (interrupted gene = the desired insert) from blue colonies (no interruption).
  • Gel Electrophoresis Principle: Separates DNA fragments by size using agarose gel.
  • Gel Electrophoresis Procedures:
    • Place gel in buffer, add DNA sample.
    • Apply electric current; DNA migrates to positive pole.
    • Smaller fragments move faster.
  • Gel Electrophoresis Staining: DNA stained for visualization under UV light.
  • Good Vector Features:
    • Small size (smaller than bacterial chromosome.)
    • High copy number (many replications per cell).
    • Multiple cloning sites (for diverse restriction enzyme cuts).
    • Selectable marker genes (e.g., antibiotic resistance, beta-galactosidase).
    • Promoter sequences.

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Description

Explore the fascinating world of recombinant DNA techniques, including the role of restriction enzymes, ligase, and DNA polymerase in manipulating genetic material. This quiz covers essential concepts such as plasmids, bacterial defense mechanisms, and the cutting of viral DNA. Test your understanding of DNA manipulation and cloning concepts!

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