(14.3) DNA Tools and Biotechnology Flashcards
How do scientists read the best sequences of DNA
By using tools that cut separate and replicate DNA base by Base
Restriction enzymes
Enzymes that cut DNA into restriction fragments (little tiny pieces of DNA u put in the plasmids)
Do restriction enzymes cut DNA at the same places
No they are cut DNA a different sequences of nucleotides
Restriction fragments
Small precise pieces of DNA only several hundred base pairs long
Gel electrophoresis
A technique used by scientist to separate and analyzed differently sized restriction fragments
What are the steps of gel electrophoresis
1) A mixture of restriction fragments is placed into wells at one end of porous gel
2) An electric voltage is applied to the gel in the negatively charged DNA moves towards the positive end of the gel
**the smaller ones move faster and the bigger ones move slower
What is the gel in gel electrophoresis Usually made of
Agarose
The ___ the DNA the faster it moves
Smaller
The wells in gel electrophoresis are located on the _____ Charged side of the gel
Negatively
There is a ____ on top of the gel in gel electrophoresis
Buffer which spreads electricity
Restriction site
Site where enzymes bond to split DNA
dideoxynucleotides
Nucleotide bases with dye in them
Chain terminating nucleotides which stop sequences when they are bonded to the sequence
Example of restriction enzyme
EcoRI
EcoRI
Restriction enzyme that recognizes the restriction sequence GAATTC and cuts between the G and A bases which leaves AATT single stranded overhangs on both strands
Sticky ends
The overhangs of AATT which can bond to a DNA strand that has complementary bases
charge of DNA
negative
plasmid
extra circular DNA in bacteria that contains accessory genes
do all bacteria have plasmids
no
SNP stands for
Single nucleotide polymorphisms
SNPs
Single base differences which are Significant because the genomes of two different individuals are identical except for about one in every 1200 bases
restriction enzymes aka
restriction endonuclease
Bio informatics
Key research area in the human genome project
New field that combines molecular biology with informational science
Genomics
The study of whole genomes including genes and their functions
how many types of DNA cloning are there and what are they
one - using bacteria
how is DNA cloning done with bacteria
so basically there’s this DNA and u chop her up with some restriction enzymes (like Ecor1) and then the SAME restriction enzymes chop up some plasmid and whoosh the DNA into the plasmids with some ligase and then the plasmids like whoosh into the bacteria and BOOM binary fission BOOM shit ton of DNA copies
cloning vector
plasmid that holds the recombinant DNA to clone it
DNA sequencing
reading the order of the bases of DNA
Dideoxy chain termination method
so basically you have a bunch of DNA strands in a little well in like those testing well thingies and then you split em up (with some HEAAATTT) and give them all the same primer and then RNA polymerase transcribes it until you get to the DIDEOXY CHAIN NUCLEOTIDE and basically transcription stops and so each time you do this (with each DNA strand) you transcribe one more base than the last time so basically when you done you just read the florescent tags and WABAM yk the order
how do flourescent tags play a role in the dideoxy chain termination method
so modern ppl basically add diff color fluorescent tags to the diff bases (A,C,G,T) so basically when you read the tags yk what base it is
why does a dideoxy chain nucleotide work
bc you cant trsnacribe past itbc there’s no oxygen so it cant do dehydration synthesis to form another phosphodiester bond
Next generation sequencing
its when have this fragment of DNA and then you add a small fragment of DNA to the end that has fluroscent tags and then you copy it a bunch of times in a well and then the computer reads it with tags
recombinant DNA
DNA from plasmid containing foreign DNA in bacteria
restriction site
the site where the recombinant DNA
sticky end
the overhangs of bases, which can bond to DNA bases
are sticky ends one base pair long
no multiple
PCR stands for
polymerase chain reaction
polymerase chain reaction
1) denature with heat and open the DNA up so u can put the primer on her
2) ANNEALING u cool her down and put the primer down
3) DNA SYNTHESIS the DNA polymerase wabooms at both strands of the DNA and makes the complementary strand and then u repeat this over and over and over again so you go from 2 double strands to 4 to 8 to 16
expression vector
basically vector that is used to carry gene of interest (plasmid)
how are eukaryotic genes cloned in plasmid
so basically there’s the gene of interest u wanna copy and Ms girl needs a primer in front of it BUT there’s a problem bc the DNA has introns and we no likey and the plasmids cant take it out cuz it can only do prokaryote type shiiiii so basically outside the plasmids the DNA turn to RNA and shes like spliced up bye bye introns and then it goes into the plasmids and the plasmids woosh into DNA with reverse transcriptase and then woosh into the plasmids with restriction enzymes and BAM electroporation into the plasmids and MULTIPLY AND MAKE GENES WORK LIKE PROTEINSYA
electroporation
the process of shocking the cell membrane to make holes in it so the plasmids can go into it
Nucleic acids probe
one strand of mRNA and a DNA that is complementary to a gene ur looking for
in situ hybridization
the process of taking the nucleic acids probe and looking for the specific gene of interest by looking for the complementary strand
microarray assays
a plate that has normal cell and weirdo cell and u take mRNA of each and you reverse transcriptase into cDNA and then you take both the cDNAs and you put em together and swish em up and then you put it on a plate with genes
the cDNA either go with a good gene or bad gene and you can look at the tags of the cDNA and if the good cDNA goes with a gene yk that gene makes good proteins, but otherwise it makes cancer proteins
in vitro mutagenesis
making mutations in a lab and then putting those cells into an organism to see what happens
how are mutations made artificially without taking cells out of the organism
with RNAi
cDNA stands for
completementary DNA
cDNA
DNA Made in a lab
reverse transcriptase polymerase chain reaction
the process of going from the mRNA to the cDNA to put it into the plasmid
RNAi stands for
RNA interference
what happens if the gene in a microarray assay pairs with both the good and bad cDNA
the gene exhibits both like good and bad
what are most of the mutations made in vitro mutagenesis
no function so js making it do nothing
CRISPR
gene editing tool for cutting DNA in certain places
how does RNAi work
siRNA is cleaved and then a protein binds to it and throws away the good DNA strand (this forms this thing called RISC [RNA induced silencing complex]) which binds to mRNA strand to silence a particular gene
CRIPSR is made of what
CAS9 and RNA guide
CRISPR cuts how
it locates and identifies sequences of genes and cuts out specific sections and then the RNA opens her up and unwinds her and then they added a little base thingie which is bad but then the DNA tries to change it but it makes a mistake and the gene js turn off and go bye
difference between rt PCR and PCR
PCR is done with DNA and uses DNA polymerase while rtPCR is done with RNA for retroviruses and uses reverse transcriptase
why does PCR occur
done so you can make a bunch of copies so you can identify the disease
