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
how do we transform bacterial cells?
-mix bacterial cells with recombinant plasmid so that bacteria take up the plasmid (transformation)
-plate cells on agar plate containing antibiotics
by what process do bacterial cells take up a plasmid from their environment?
transformation
what is a colony?
many cells arising from the same cell
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 primers used in PCR?
short pieces of single stranded DNA that are complementary to either end of the gene that you wish to amplify
what is a genome
total DNA in a cell
what is a genomic library
-collection of DNA fragments of the entire genome produced by restriction digest
-each fragment is cloned into a plasmid
-like reserves of DNA we keep in a freezer
what are the sources of DNA for cloning?
-genomic library
-cDNA
how is cDNA made?
-mRNAs (mature) are isolated from a cell
-reverse transcriptase enzyme is used to make a complementary strand
-DNA polymerase synthesizes the second DNA strand to make a double stranded cDNA molecule
-cDNA molecules are cloned into plasmids
does cDNA have introns?
no, because it is from the cytoplasm, therefore it is mature mRNA and has already been spliced
what are the reasons why eukaryotic genes are not always expressed well in prokaryotic cells?
are control elements introns or exons
introns
true or false, when genomic DNA from different individuals is digested with the same restriction enzymes, the pattern of DNA fragments produced is the same
false
what is the difference in the pattern of DNA fragments between two people with different genomic libraries cut with the same restriction enzyme due to?
single nucleotide polymorphisms (SNPs) scattered throughout the genome
what is an SNP?
a genomic variant at a single base position in the DNA
what do SNPs cause?
restriction fragment length polymorphisms (RFLP) between individuals due to the fact that they prevent the cutting at certain restriction sites
what are the applications of restriction fragment analysis?
DNA fingerprinting, distinguishing between normal and disease allele,
what can DNA fingerprinting be used for?
to determine the paternity (RFLPs are inherited in a mendelian way)
how can restriction fragment analysis help us distinguish if we have a normal or disease allele?
since they’re gonna be cut different, the electrophoresis of fragments will look different
true or false, even SNPs next to a gene can be used to differentiate between a normal and a disease-causing allele
true
what do u need for dideoxycytidine’s chain termination method?
-DNA to sequence (template); DNA is denatured
-primer
-DNA polymerase
-Deoxyribonucleotides
-dideoxyribonucleotides
mix everything together!
what are dideoxyribonucleotides?
modified dNTPs that act as chain terminators
what are the colours of the dideoxyribonucleotides? how many are there?
each of the 4 ddNTPs is tagged with different colour fluorescent tag
purple: ddATP
pink: ddCTP
yellow: ddTTP
green: ddGTP
each chain terminator is a base (adenine, cytosine, thymine, guanine)
what is the point of DNA sequencing with dideoxy chain termination method?
determine the DNA sequence of the DNA sequence
how does the dideoxy chain termination method help us determine the sequence
each colour is associated to a base pair, and the number of base pairs in the strand show us the position of the base pair (since its a termination, we know its the last one in the sequence)
what are the 3 steps to DNA sequencing?
step 1: mix DNA, primer, DNA polymerase, deoxyribonucleotides, dideoxyribonucleotides together
step 2: DNA polymerase synthesizes a complementary DNA strand to the template
step 3: DNA fragments are separated by gel electrophoresis
what happens during step 2 of DNA sequencing?
-DNA synthesis of a strand will be terminated as soon as a ddNTP is incorporated
-DNA fragment will be fluorescently labeled and colour of fluorescence depends on which ddNTP terminated the strand
-there will be fragments of every possible length
what happens during step 3 of DNA sequencing?
-As DNA fragments move through the gel they pass through a fluorescence detector
-the colour of the fluorescence detected tells you which base is in each position
in which fragment size order do fragment sizes move past the fluorescence detector? why?
from smallest to largest cause smaller fragments move faster in gel electrophoresis
