biotechnology

what is the process of producing recombinant dna? (7 steps)

1. desired gene identified and isolated

2. restriction enzyme used to extract gene of interest from donor organism at specific recognition site

3. same restriction enzyme used to cut bacterial plasmid so both pieces of dna have complementary sticky ends

4. sticky ends joined through hydrogen bonding between complementary bases

5. dna ligase used to seal sugar phosphate backbone

6. recombinant dna inserted into host cell/vector dna

7. transformation occurs when recombinant dna introduced into host cell by heat shock for cloning and gene expression

why can we combine dna from at least two organisms of different species successfully?

dna is universal so enzymes can be used for all dna molecules no matter where dna originates from

what is the difference between a transgenic organism and a gmo?

a gmo is an organism whose genome has been altered using laboratory techniques while a transgenic organism is a type of gmo that contains foreign dna from a different species

what is the process of creating a transgenic crop using recombinant dna? (4 steps)

1. transfer recombinant dna into plant cells using Agrobacterium tumefaciens vector

2. select successfully transformed cells using suitable marker

3. grow transformed cells into whole plant using plant tissue culture

4. test plants to confirm foreign gene is expressed

what is the process of creating a useful protein using recombinant dna?

1. insert recombinant plasmid into bacterial cells through transformation

2. select bacteria that have successfully taken up plasmid

3. culture bacteria under ideal conditions allowing it to reproduce and express gene

4. harvest and purify protein produced by bacteria

what is the purpose of pcr?

to amplify specific sequence of dna

what is taq polymerase?

heat stable form of dna polymerase from thermophilic bacteria thermus aquaticus

what happens in the denaturation step of pcr?

reaction heated to 94ºC to break hydrogen bonds between bases. double stranded template dna separates into two single strands

what happens in the annealing stage of pcr?

reaction cooled to 50-65ºC so primers can bind to complementary sequences on template strand

what happens in the extension stage of pcr?

reaction heated to 72ºC to allow taq polymerase to attach free complementary nucleotides to build new complementary dna strand

what is gel electrophoresis?

technique used to separate dna fragments according to size using electric field

what is the process of gel electrophoresis? (5 steps)

1. agarose gel placed into container filled with buffer solution. gel has small pores

2. dna sample placed into small wells at one end of gel

3. electric current passed over gel and negatively charged dna migrates towards positive electrode

4. smaller fragments move through pores more easily and travel faster and further. larger fragments face more resistance and move slowly and stay closer to wells

5. scientists use dna binding stain and imaging method to make dna fragments visible as bands