Chapter 20-21 Biotechnology and Genomics Flashcards
What is genetic engineering?
extraction and reconfiguration of DNA
What is the goal of genetic engineering?
to impact the nucleic acid by recombing DNA
What is recombinant DNA?
taking DNA from different locations and putting it into one nucleic acid molecule
What is a natural example of recombinant DNA?
crossing over and viruses
Which areas can genetic engineering be applied to?
medicine, pharmacy, environmental, forensic analysis, and agriculture
What are restriction enzymes?
enzymes floating around recognizing original DNA and destroying foreign DNA
What do restriction enzymes need in order to work?
a specific sequence that is from the bacterial cell
What are the sequences that restriction enzymes recognize called?
palindromic sequences
What do restriction enzymes do the recognizable DNA?
cleaves the DNA to make two strands
After restriction enzymes cleave the DNA, what is created?
two strands with sticky ends
What are sticky ends?
float around and recombine/reanneal with their complementary strands
What are restriction enzymes used to make for humans?
insulin
How do restriction enzymes make insulin?
restriction enzymes cut the bacteria DNA, then attach the human insulin gene via a vector on the sticky ends and combine the DNA so bacteria synthesize insulin
What are polymorphisms?
Variations in DNA sequence are called
polymorphisms
What are RFLPs
restriction fragment length
polymorphisms — a type of polymorphism that results from variation in the DNA sequence recognized by restriction enzymes.
What is gel electrophoresis?
separates molecules based on their size by the use of a gel matrix and electric currents
What can gel electrophoresis be used on?
DNA, RNA, proteins
How is DNA used in gel electrophoresis?
since DNA has a negative charge on the phosphate, they are placed in the holes (aka wells) on one end of the gel and a negative current pushes the DNA to the positive side
What is the gel on gel electrophoresis?
agarose (polysaccharide polymer)
How are restriction enzymes and gel electrophoresis related?
comparing DNA of organisms requires the same restriction enzymes to cut the same segment of DNA from both organisms to compare at identification points
In the gel electrophoresis, what do different band lengths signify under UV light?
shorter DNA moves faster and towards the other end of the gel and longer DNA is more towards the wells
Why is gel electrophoresis important?
helps determine relatedness in different species and organisms, DNA fingerprinting
What is Polymerase Chain Reaction (PCR)?
helps produce many copies of a
the specific target segment of DNA
What do you need for PCR?
the DNA portion to copy, a buffer to put it in, primers, DNA polymerase, DNA nucleotides
Does PCR have to happen in a cell?
NOPE, it can happen in a test tube
What kind of DNA polymerase is used for PCR?
heat resistant Taq polymerase
Where is the Taq polymerase from?
from bacteria that originally lived at boiling temperatures
What is the first step of PCR?
heat up DNA to separate the strands (acts like helicase)
What is the second step of PCR?
annealing involves designing primers that specify the target sequence and having those primers hydrogen bond to target at a certain temp
What is the third step of PCR?
DNA synthesis –> make more copies of DNA using DNA polymerase using DNA nucleotides at a certain temp
Why is PCR important?
DNA fingerprinting and disease diagnosis (covid-19)
Single genes are cloned using what?
plasmids in bacterial cells
What are plasmids?
small, independent, and circular mobile pieces of DNA that contain nonessential genes
Are plasmids a part of the bacterial genome?
no
What are vectors?
DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell
How does single gene cloning work?
put gene on vector and insert into plasmid then put back into the cell to create more copies of plasmids
What is cloning organisms also called?
reproductive cloning
What does cloning use?
nuclear transplantation
What is nuclear transplantation?
the nucleus of an unfertilized egg cell is replaced with the nucleus of a differentiated cell from an adult organism
What can vectors lead to?
GMOs (the first GMO food was a flavor savor tomato)
How did cloning animals work? aka Dolly
normal adult nucleus taken from the udder of sheep 1, DNA extracted, egg cell from sheep 2 had sheep DNA put into it and was put into an embryo
What are stem cells?
an unspecialized cell that can reproduce indefinitely and can differentiate into specialized cells
How do you isolate stem cells?
can be isolated from early embryos at the blastocyst stage (ball that is hollow, stem cells on the inside)
Where are stem cells found in adult humans?
bone marrow, but they only form blood related cells
For stem cells, what determines what cell type it will become?
transcription factors
What is a totipotent stem cell?
can divide into all cell types in an organism
What is a pluripotent stem cell?
can divide into most, or all, cell types in an organism, but cannot develop into an entire organism on their own
What are IPS cells?
artificial pluripotent cells that have been developed by making a specialized cel become a stem cell again via 4 types of enzymes/viruses
What is CRISPR-Cas9?
Clustered Regularly Interspaced Short
Palindromic Repeats
What does CRISPR-Cas9 use?
uses short segments of DNA that are palindromic (sequence of letters that read the same from left to right) that has spacer DNA in between the segments
What is the spacer DNA in CRISPR-Cas9?
not identical DNA (unique) that matches with viral DNA and cas genes associated with helicase and nucleases
What does CRISPR-Cas9 act as?
a tiny immune system
When the virus injects their DNA into the bacteria, what happens in CRISPR-Cas9?
CRISPR complex transcribes proteins w/ crRNA/gRNA and the cas proteins break the viral DNA up and embedded it into the CRISPR sequence
What does the cas9 protein do?
cuts DNA using CRISPR RNA (gRNA) to inactivate the gene and even mutate it
What is the first generation method for genomic sequencing?
Sanger Method
What type of nucleotide is used by the sanger method?
dideoxyNTP (ddNTP)
What is a ddNTP?
nucleotide that DOESN’T have any oxygen on 3’
What does 3’ position on a nucleotide determine?
its the backbone to where nucleotides in the sequence can link up to where OH usually is
What is ddNTP used for?
termination of synthesis of DNA in the Sager method of DNA sequencing
What mechanism is the Sanger method based on?
DNA synthesis/replication
What do you need for the Sanger method?
unknown template strand copies of DNA from 3’ to 5, primers that are fluorescently labeled, DNA polym, dNTP (large amount), and ddNTP (small amount)
What is the Sanger method also called?
chain termination method
What are ddNTPs called?
chain terminators
What happens in the Sanger method?
separate dNTPs put into 4 test tubes, put ddNTP into it
What happens when ddNTP is added to the test tubes?
number fragments of copied DNA that is terminated at certain base pairs (ex: TAAGTCCCT turns to A, TAAGA, TAAGTCCA) according to how many nucleotides are there
What is the result of the Sanger method?
DNA fragments with various lengths
What happens to the DNA fragments in the Sanger method?
put into gel electrophoresis to separate the lengths of the DNA fragments, read from smallest to largest
What is the second generation of DNA sequencing?
NGS (next generation sequencing)
What happens in NGS?
short-read sequences from about 50 base pairs to 300 base pairs
What is the gold standard for gene sequencing?
sanger method
What is the first step in NGS?
DNA gets fragmented and replicated with adaptors on each end of the fragment
What is the second step of NGS?
fragments get put onto a glass slide with sequences that are complementary to the adaptors so that the fragments go near the complimentary base pair
What is the third step of NGS?
make copies of DNA fragments using polymerase on the slide in the local region and add fluorescent dNTPs
What is the fourth step of NGS?
as the sequence is getting synthesized, computer reads fragment as it is getting synthesized and aligns the overlapping fragments to make one genome
What is the 3rd generation of genetic sequencing?
nanopore and smrt
What happens in nanopore 3rd generation of sequencing?
DNA is fed into an artificial membrane and electric current is sent through the membrane, the nucleotides are disrupted at a specific voltage
What happens in SMRT 3rd generation sequencing?
DNA broken into fragments, fluorescent added to the end of phosphate, put into little wells, DNA polymerase lights up the different nucleotides
What is genomics?
study of whole sets of genes, structure, function, evolution, and mapping of genomes
How much of the DNA is used for the coding sequence?
2 percent
What is intergenic DNA?
rest of the DNA that is not coding and is found between genes
What are the genetic markers?
single nucleotide polymorphism and short tandem repeats
What are SNPs?
one base-pair track which is found in the coding region
What are STRs?
little sequence repeats that differ from person to person
What are transposable elements?
makeup 45% of the human genome and they are genes that are able to jump to another position on the genome
What are the types of transposons?
DNA transposon and retrotransposon
What are DNA transposons (eukaryotes)?
transposase cuts and pastes gene from point to point b
What are retrotransposons (3 classes)?
use of reverse transcriptase to copy and paste so that genome can increase in size and change profiles
What are human’s cousins?
chimp that has 24 chromosomes instead of 23 (humans had a fusion)
Where do chromosomal changes happen?
crossing over in meiosis for recombination
How does divergence happen?
cells deliver different chromosomes to isolate organisms
How do changes to chromosomes help speciation?
creates new species that are able to adapt to the environment
What are some types of chromosomal arrangements?
can delete or duplicate regions of a chromosome when similar regions of genes are crossed over at the wrong location to create multi family genes
What is an example of multifamily genes?
the globin genes because there is alpha and beta
What did the globin genes diverge from?
their ancestral globin gene (400-450 mill years ago)
When are the globin genes expressed?
during embryonic, fetal, and adult stages of development
What are the types of globin genes?
alpha and beta (polypeptides)
What are pseudogenes?
genes that don’t work anymore and are leftover from evolution due to mutations
How are genes with new functions created?
in exon shuffling
What happens in exon shuffling?
mistakes in meiosis can duplicate or reposition exons in a more permanent
What is an example of exon shuffling?
tpa gene (blood clotting)
What are palindromes?
sequences of DNA that are repeated and spaced
What do the cas genes have?
helicases and nucleases (that cut DNA)
Where do restriction enzymes bind to?
restriction sites (DNA sequence)
