Topic 10: DNA Technology and Applications in Genetics Flashcards
what are the three new technologies for molecular genetics?
polymerase chain reaction (PCR)
DNA sequencing (snager method, next generation, third generation)
Restriction enzymes
who developed PCR in 1983?
Kary Mullis
what are the three main steps of PCR?
- DNA denaturation (1-2 mins)
- Primer annealing (1-2 mins)
- Polymerase extension (3-5 mins)
to what temperature do you heat the reaction mixture to denature DNA in PCR?
95 degrees Celcius
PCR produces DNA fragments of varying lengths, how can we visualize these fragments?
gel electrophoresis
separates DNA fragments by their base pair lengths, longer fragments are heavier and move slower than smaller fragments
di-deoxynucleotides are like dNTPs but lack two oxygen, what is this then abbreviated to be?
ddNTPs
what is the official scientific name for the “Sanger method of DNA sequencing?
dideoxynucleotide DNA sequencing
true/false: lack of OH prevents the ddNTP from forming phosphodiester bonds
true
true/false: in dideoxynucleotide DNA sequencing, 5-3’ polarity corresponds to the smaller-to-larger fragment length direction
true
how does next-generation DNA sequencing (NGS) work? why is it faster and cheaper than the Sanger method
uses computational power to put fragments together into genome-wide consensus sequence
true/false: third-generation DNA sequencing (TGS) isn’t very similar and parallel to next-generation DNA sequencing
false, its very similar and parallel
restriction enzymes are a form of __________
recombinant DNA technology
recombinant DNA technology amplifies, maintains and ________ specific DNA sequences inside the cell
manipulates
_____________: uses specific enzymes to cut DNA at specific points resulting in double-stranded breaks
restriction mapping
true/false: if two DNA molecules have sticky ends produced by the same restriction enzymes, they can be joined together
true
_________: stick to complementary base-pair sequence
sticky ends
what are the main steps of restriction mapping?
- digest DNA with restriction enzymes (cut)
- separate fragments with electrophoresis
- visualize DNA
_______: is a carrier fragment of DNA
vector
molecular cloning is a technique that inserts restriction fragments into a vector, why do we do this?
when we want to study what a gene actually DOES
a vector is inserted unto an organism (usually bacteria), which then amplifies the _____________ within the cell, making _________
recombinant DNA molecule
DNA clones
what kind of forensic analysis can be done through genetic techniques?
DNA fingerprinting/DNA profiling
what does DNA fingerprinting/DNA profiling use to match people to DNA?
short tandem repeats (STR)
what are the four criteria of the combined DNA index system?
- must have a known chromosome location that independently assorts from all other CODIS STR’s
- must have multiple alleles per STR locus in all populations examined
- STR alleles must be consistently, reliably, and accurately able to be amplified via PCR
- PCR products must distinguish alleles from each other clearly enough for the automated PCR amplification and gel electrophoresis to reliably ID each allele
in paternity testing, a _______ is calculated for each gene tested (x/y, the probability that a trait actually came from the father in question)
paternity index (PI)
all paternity index’s are added up to be a __________, which represents all genes analyzed
combined paternity index (CPI)
________: changing the nucleotide sequence at a specific chromosomal locus to any desired sequence
gene editing
how does gene editing work? what does it use?
uses a DNA endonuclease to cleave the DNA at specific target locations within the genome ( results in double-stranded breaks, which allows the genes to be modified)
what are the two main DNA repair mechanisms?
non-homologous end joining
homology-directed repair
________ allows for gene knockouts/knockins
non-homologous end joining
how does non-homologous end joining work?
proteins recognize double-stranded breaks, the protein complex attaches to both broken ends, the ends are trimmed until a blunt end is achieved, DNA ligase glues the blunt ends together
we do lose some nucleotides from the trimming of the DNA
_________ allows for gene insertions and/or modifications
homology-directed repair
how does homology-directed repair work?
- a double-stranded break occurs
- nucleases trim off a portion of the broken strand, Rad51 binds the undamaged chromatid
- strand invasion occurs in a displacement loop (unbroken and broken chromatids are woven together)
- DNA replication within the D loop synthesizes new DNA using the sister chromatid as a template
- DNA strands are trimmed to blunt ends ad are ligated to finish repair
true/false: gene editing tools exploit the cell’s natural DNA repair mechanisms
true!
what are the two ways artificial gene editing can exploit the cells natural repair mechanisms? where do they “attack”?
DNA binding domain
incorporated into a complex with an RNA molecule
what were the first two gene editing tools called?
ZFNs and TALENs
true/false: CRISPR/Cas9 was first found in eukaryotes
false, it was first found in prokaryotes in the 1990s
what does CRISPR stand for?
Clustered Regularly Interspersed Short Palindromic Repeats
what is the official name for CRISPR-Cas9
CRISPR-associated (cas) genes
CRISPR/Cas9 is a natural defense mechanism in prokaryotes
1. CRISPR sequences are transcribed into non-coding RNA\
2. The tracer RNA gene is also transcribed into ________ and Cas9 is produced
3. Processed into _______ by cas-encoded Rnasas
tracrRNA
crRNAs
true/false: tracrRNA and crRNA form a complex with CAS endonucleases to cleave foreign DNA
true
true/false: the prokaryote can store the cleaved DNA in its genome for future invader recognition or inheritance
true, can incorporate it into its CRISPR region
true/false: CRISPR can’t be passed down to progeny
false! it can!!
how have scientists engineered CRISPR to target any sequence of interest? what have they binded together?
crRNA and tracrRNA are fused together into single guide RNA (sgRNA), which scientists can then insert their target DNA into, creating a unique crRNA
Cas9 is used as the endonuclease
once Cas9 makes its cut, cell DNA repair finishes gene editing, how do the two DNA repair mechanisms do this?
homology-directed repair:
uses supplied DNA fragments as the template for DNA synthesis
nonhomologous end joining:
either results in the deletions of one or more nucleotides, but can also introduce insertions of DNA derived from elsewhere in the genome
what three domains make up the Cas9 endonuclease?
HNH domain, RuvC domain, PAM
what does the HNH domain do?
cleaves the strand complementary to the crRNA
what does the RuvC domain do?
cleaves the opposite strand of DNA
what does PAM do?
protospacer adjacent motif, destabilises the adjacent sequences for crRNA complementary pairing (in Cas9=NGG)
what are the three ways to get CRISPR DNA into cells?
mediated transformation, infiltration, particle bombardment
agrobacterium contains a plasmid (DNA) called ____________ (Ti plasmid)
tumor-inducing plasmid, a portion of the plasmid is called transfer DNA (T-DNA), which can transfer to the plant and recombine with the host genome
agrobacterium-mediated transformation:
- the _____ of the Ti plasmid is transcribed
- Virulence proteins are transcribed and translated
- Conjugative transfer proteins are transcribed and translated
- The T-DNA will insert itself randomly into the nuclear genome
T-DNA
how is a Ti-plasmid disarmed? to transfer any gene of interest to plants
by removing the opine- and tumor-inducing genes from the T-DNA
when transferring a gene of interest to a plant, a gene of interest is inserted between the left and right T-DNA birders into a ________________ (a second plasmid)
transformation vector
true/false: when transferring a gene of interest to a plant, the two plasmids formed are re-inserted back into the agrobacterium they came from
true
true/false: genes on the disarmed plasmid are transcribed and translated to virulence and conjugation proteins
true
true/false: the T-DNA is transcribed from the transformation vector and inserted into the plant host nuclear genome
true
CRISPR/Cas9 is extensively used in plant biology research, most edits are via _________ repair to promote gene knockouts/downs to determine gene function in model plants
NHEJ (nonhomologous end joining)
_____________(GMO’s) are defined as organisms that have had their DNA changed by artificial means
genetically modified organisms
why do some countries allow GMO’s? what is the condition that must be met?
they are allowed as long as the results COULD’VE been achieved via selective breeding (i.e we could’ve naturally done it with lots of time and effort, no foreign DNA introduced)
