recombinant DNA technology Flashcards
what does PCR stand for?
polymerase chain reaction
why is PCR amazing?
-quick amplification of DNA (within a few hours)
-highly specific
-only small amount of DNA is required
why is PCR said to be highly specific?
-primers are designed to target a specific DNA sequence within an entire genome
what are a few applications of PCR?
-cloning genes
-covid test
-crime scene analysis
-amplifying sources of DNA when there’s very little starting material
what do you need to conduct a PCR test?
-DNA (contains sequence you want to amplify)
-primers (short pieces of single stranded DNA that are complementary to either end of the gene that you wish to amplify
-nucleotides (dNTPS)
-DNA polymerase (Taq)
what is Taq?
thermostable DNA polymerase (does not denature at 95 degree Celsius)
what are dNTPS?
deoxyribonucleotides. can be either adenine, guanine, cystine, etc.
why do we need primers to do PCR?
the same reason we needed primers for DNA replication in the earlier chapters. DNA polymerase can only extend from a free 3’ end.
what are the PCR steps?
1) denaturation: heat tube to 95 degrees celcius to denature DNA (unzip DNA)
2) annealing: cool to 55 degrees Celsius to allow the primers to bind to the DNA
3) extension/polymerization: heat to 72 degrees Celsius for Taq DNA polymerase to synthesize complementary DNA
repeat cycle over and over for exponential amplification!
after how many cycles of DNA replication is the desired DNA fragment isolated? how many fully replicated fragments do we have?
after cycle 3, we have 2 molecules that match the target sequence exactly (same length and match target sequence)
what is the definition of gene cloning?
making multiple copies of a gene
what are the purposes of gene cloning?
1) produce a protein (insulin, growth hormone)
2) produce copies of a gene (ex: gene for pest resistance inserted into a plant)
what are the tools for gene cloning?
1) plasmid
2) gene of interest
3) restriction endonucleases
why do we need restriction endonucleases in cloning?
-hydrolyze phosphodiester bonds
-cut DNA st specific restriction sites
-both strands are cut to produce overhangs called “sticky ends”
in gene cloning, how is the gene of interest amplified?
by PCR
in gene cloning, what is the source of the gene of interest?
genomic DNA or cDNA
what are the steps to cloning?
1) isolate plasmid from bacteria and isolate DNA containing gene of interest
2)amplify gene of interest using PCR (primers are added when we do PCR, and primers contain restriction site)
3) cut plasmid and amplified gene of interest with same restriction enzyme
4) mix cut DNA together and add DNA ligase to produce recombinant plasmids
5) transform bacteria with recombinant plasmids and select for transformed bacteria using antibiotic resistance
why do we want to cut plasmid and amplified DNA with same restriction enzyme?
if the plasmid and the genomic DNA are cut with the same restriction enzyme, they will have complementary sticky ends
how do we create a recombinant plasmid?
-mix cut plasmid and cut gene of interest together
-complementary sticky ends will base pair
-add DNA ligase to “glue” strands together
why do we put the cells that we mixed with the recombinant plasmid on an antibiotic plate?
because we only want to keep the cells that acquired the plasmid. since the plasmid contains an antibiotic resistant gene, the cells that contain the plasmid will continue living, and the ones that dont will die. we only want the cells that contain the plasmid so tis is useful to us
what are the reasons why eukaryotic genes are not always expressed well in prokaryotic cells?
