Biotechnology Techniques

Recombinant DNA: DNA made by connecting DNA from two different sources.

Example: a human insulingene inserted into bacterialDNA.

Organisms containing recombinant DNA are called transgenic.

Producing Recombinant DNA

Utilizes Utilizes restriction restriction enzymes: enzymes: enzymes that cut specific enzymes that cut specific sequences of DNA.sequences of DNA.

The way that restriction The way that restriction enzymes cut DNA allows enzymes cut DNA allows DNA from 2 different DNA from 2 different sources to be joined sources to be joined together.together.

Sticky EndsSticky Ends

Many restriction enzymes cut Many restriction enzymes cut sequence in a way that produces sequence in a way that produces single-strandedsingle-stranded “sticky ends”. “sticky ends”.

Any two DNA strands cut with the Any two DNA strands cut with the SAME restriction enzyme can bind SAME restriction enzyme can bind together due to complementary together due to complementary pairing of sticky ends.pairing of sticky ends.

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Complementary sticky ends allow the DNA

from 2 different

sources to join together.

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Discuss with your table partner:Discuss with your table partner:The definition of recombinant DNAThe definition of recombinant DNAHow scientist are able to join two How scientist are able to join two pieces of DNA from different sources pieces of DNA from different sources togethertogetherSome uses of recombinant DNA Some uses of recombinant DNA technologytechnology

Vectors Vectors are a means by which foreign

DNA can be transferred into a host cell.

Common vectors: Plasmids (small circular secondary

chromosomes from bacteria) Viral DNA

Bacterial Transformation

Once a transgenic plasmid is produced, it can enter a bacterial cell under certain conditions. This uptake of foreign DNA by bacteria cells is called transformation.

Plasmid Video: Click once on image to start video

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Conditions for Transformation: Treatment with CaCl2 makes the bacterial cell wall more

permeable and help to allow the uptake of the foreign DNA.

Screening for Successive Transformation

Not all the bacterial cells will successfully take in the foreign DNA with the desired gene.

In order to be able to screen for successfully transformed cells, scientists often chose a plasmid vector with an antibiotic resistance gene and a host cell that is susceptible to the antibiotic.

Transformed bacteria cell with antibiotic resistance gene on

plasmid

Bacteria cell that did NOT receive plasmid

Will be able to grow in the presence of the

antibiotic as well as on regular petri dishes.

Will only be able to grow on petri dishes

that do NOT contain the antibiotic.

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Discuss with your table partner:Discuss with your table partner:

The definition of transformation (for The definition of transformation (for bacteria)bacteria)Screening of bacteria for successful Screening of bacteria for successful transformationtransformation

PCR:Polymerase Chain Reaction

Magnifies minute quantities of DNA so that Magnifies minute quantities of DNA so that they can be analyzed.they can be analyzed.

Process:Process: DNA heated to separate strands (breaks DNA heated to separate strands (breaks

weaker hydrogen bonds between nitrogen weaker hydrogen bonds between nitrogen base pairs).base pairs).

Single strands incubated with DNA Single strands incubated with DNA polymerase and free nucleotides to replicate.polymerase and free nucleotides to replicate.

Process repeated multiple times.Process repeated multiple times.

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Review with your table partner the Review with your table partner the process of polymerase chain reaction process of polymerase chain reaction (PCR)(PCR)

What is the purpose of PCRWhat is the purpose of PCRWhat is the basic processWhat is the basic processHow does the structure of DNA relate How does the structure of DNA relate to how this process works (Hydrogen to how this process works (Hydrogen bonds, base pairs)bonds, base pairs)