ch 20 Flashcards
(87 cards)
1
Q
cloning vector
A
a modified plasmid or virus used to transfer recombinant dna
2
Q
restriction endonuclease/restriction enzyme
A
- enzyme from bacteria that cut dna at a specific basepair sequence called the recognition site
3
Q
dna cloning involves
A
1) restriction endonuclease cuts dna at recognition site
2) dna ligase seals the dna together
4
Q
gene knockout
A
engineering a gene to lose function, understand function of gene by seeing effect of when it doesn’t work
5
Q
how many genes do mice and humans have significant similarity?
A
16000
6
Q
gmos and development
A
1) add a short sequence of dna to a gene that codes an easily detected protein (often one that is fluorescent)
2) when recombinant gene is expressed, animal lights up so we know when that part of development happens
7
Q
transgenic
A
organism whose genome contains dna from another organism
8
Q
Ti plasmids
A
1) a bacterium with T-dna in the Ti plasmid infects cells to incorporate dna
2) when Ti is transcribed, plant cell division is induced
9
Q
gmo Ti plasmids
A
1) tumor-inducing regions in T-dna region of Ti plasmid removed
2) replace with desired dna
3) new genes now in plant’s genome
10
Q
how can a gmo plant be made from a single cell?
A
1) cell with other organism’s gene added to nutrient-rich medium
2) mass of cells grow (a callus)
3) transfer callus to medium that promotes plant development
4) seeds from it used to create more gmo plants
11
Q
successful gene therapy requirements
A
1) disease must be due to defects of a single gene
2) sequence of wildtype allele is known
3) allele must be introduced into correct tissues at correct amount and correct time
4) for dominant disease alleles have to be able to replace defective allele with functioning allele (for recessive you can just add a functioning allele)
12
Q
CAR-T cells
A
- gmo immune system cells to recognize tumor cells
- engineered outside of patient’s body and infused to destroy blood cancer cell
- ex vivo
13
Q
vectors
A
- genetically engineered viruses
- allow incorporation of therapeutic genes
- disable target virus replication
- must be able to gain access to cell
14
Q
ex vivo
A
cells needing therapeutic gene removed and infected w/ viral gene therapy vector, typically integrate therapeutic gene into genome
15
Q
in vivo
A
viral gene vector injected into bloodstream
16
Q
hemoglobin throughout development
A
- different forms of hemoglobin produced at different stages of human development, each form prod by diff genes
- ALL forms of hemoglobin function well enough to carry O2 at any dev stage, but each stage has an optimal form
17
Q
CRISPR-Cas discovery
A
- discovered in salt-tolerant archaeon
- found short rep dna sequences separated by spacer sequences w no similarity to each other or rest of genome
- widespread phenomena in bacteria and archaea
- these spacers were from viruses, which makes these organisms more immune to those viruses in the future
18
Q
CRISPR-Cas gene locus (how it works)
A
- transcribed into long pre-crRNA
- processed into shorter crRNA fragments that contain an RNA copy of part of a viral genome
- when a virus invades, crRNA recognizes it and binds to it
- crRNAs are base paired at one end with tracrRNA (noncoding, stands for trans-activating)
- crRNAs and tracrRNAs are bound by Cas protein, an enzyme that CUTS complementary dna
19
Q
how common is the CRISPR-Cas system?
A
- found in 80% of archaea and 40% of bacteria
- at least 6 diff types
20
Q
in vitro
A
created outside of a living organism, such as in a tube or petri dish (think vitro=glass in Latin)
21
Q
sgRNA
A
single guide RNA, a fusion of crRNA and tracrRNA, makes genome editing possible
22
Q
how does crispr-cas cut dna
A
1) mix sgRNA with plasmid DNA that contains cas9 gene
2) add this nucleic acid mixture to cell
3) when cas9 is expressed it will associate w sgRNA and bind to complementary target sequence
3) cas9 endonuclease makes double stranded cuts in dna
23
Q
how is genome edited after being cut by crispr-cas
A
- dna repair mechanisms: NHEJ or HDR
24
Q
NHEJ (nonhomologous end joining)
A
- insertions or deletions i-inactivate target gene
- destroys genes
25
HDR (homology-directed repair)
- cut site recombines w/ engineered dna gene, inserted at target locus precisely
- this dna fragment must also be added with crispr-cas for this to work
- modifies/replaces genes
26
crispr now
now we can:
- alter specific base pairs w/o cutting
- reg transcript of specific genes
- add/remove epigenetic mods
- mod multiple genes simultaneously
- tag genes w fluorescent molecs
- screen for gene function of every possible genome
- target and destry specific rnas
27
gene drive
- crispr-cas is used to spread a particular form of a gene throughout pop in only a few gen
- causes concern if gmo organism enters wild
28
gene drive locus
- a modified gene that contains sequences for sgRNA and cas9
- allows gene to spread rapidly through pop
- organisms quickly become homozygous for this allele
29
pcr
- allows quick gene cloning (possibly in hours)
- amplifies a specific sequence of dna
- requires primers that match sequences on either side of amplified sequence
30
things possible bc of pcr
- dna fingerprinting
- paternity testing
- testing for multiple bacterial/viral pathogens
-analyzing ancient dna
- dna-based genealogy
31
pcr steps
1) denaturation
2) annealing
3) extension
- repeated in a cycle, allows for exponential amplification of dna
- depends on separating complementary dna strands at each amplification cycle
32
dna fingerprinting
- identifying individuals by unique features in their genome
33
tandem repeats
- thousands of sites that contain short sequences of dna that are repped one after another along part of a chromosome
- repeat number varies btwn individuals
34
STRs (short tandem repeats/microsatellites/SSRs [simple sequence repeats])
- often used in modern dna fingerprinting
- consist of simple repping units 2-8 nucleotides long
- non-coding regions--no effect on phenotype
- proposed origin: when dna polymerase skips or adds extra bases during replication by mistake
- use gel electrophoresis to see
- the shorter the pcr product, the farther down the gel it will be
35
eDNA (environmental dna)
- tells us which species exist in an environment and relative abundance of species
- can detect species that no one's observed
- used in forensic science as well
- organisms leave dna everywhere
36
dideoxy sequencing/sanger method
- normal dNTPs with chemically modified ddNTPs (which has no 3' hydroxyl group)
- ddNTPs stop synthesis
- sequence of dna determined by detecting the terminal 3' base (A C G or T) at each position in the DNA
- reads about 750 bases reliably
37
denaturation pcr
separates the two strands of dna (i think at extremely high temps)
38
annealing pcr
attach primers to dna
39
extension pcr
synthesize complementary dna (cDNA) from dNTPS, starting at primer
40
NGS (next-generation sequencing)
- rapidly determine entire sequence of genome
- faster and cheaper than dideoxy sequencing
- millions of diff dna can be amplified and sequenced in single run
41
shotgun sequencing
- many copies of genome broken up into random diff-sized fragments
- fragments expected to overlap, which guides putting the whole genome back together (genome assembly)
42
genomic dna
derived directly from cells, not complementary
43
reference genome
first high quality sequence obtained by a given species becomes the standard for comparing and analyzing other individuals from that species
44
how many known genome sequences are there?
~350k, including 5.5k animal and 1.7 plant
45
bioinformatics
math, comp sci, and bio combined to manage and analyze sequence data, used to evaluate similarities btwn genes and genomes
46
genome annotation
identifying which regions of the genome contain genes/functionally important sequences
47
how many potential reading frames on an rna strand?
3, so 6 on the two rnas that would come from a double stranded dna
48
how many stop codons on average
1 in every 20 codons
49
ORF (open reading frame)
- indicator of protein coding sequence, long stretch of codons w/o stop codon
- when found, is compared with known sequences from other species--> if unlike any other species, will be further researched, is similar, it's likely a gene
50
ESTs (expressed sequence tags)
- a complentary dna (cDNA) from part of a transcribed gene used to find physical location of gene in the genome to confirm that a region of dna is transcribed
- if a cDNA can be made of even part of an rna, then these regions can be searched for in the genome and are likely genes
51
GWAS (genome-wide association study)
- locating genes by finding associations btwn a phenotype and a polymorphic genetic marker (almost always an SNP) in populations
- ex: comparing to find common allele pair present in diabetics but not in non-diabetics
- look for coinheritance btwn phenotype of interest and dna sequences at known locations in genome (genetic markers)
52
genetic markers
genetic locus that can be identified and traced in populations by phenotype or molecular testing
53
SNP (single nucleotide polymorphism)
- a site in dna that varies by a single base pair
- if trait and snp allele (in GWAS) almost always occur together, then the gene for the trait must be close to the SNP (think: genes close together will be inherited together)
- most are noncoding and don't effect phenotype
54
lateral gene transfer
- transfer of dna btwn two diff species aka horizontal gene transfer
- a significant proportion of many prokaryotic genomes have been acquired from other distantly related species
- major force in evolution of prokaryotes
- sometimes happens bc of plasmids and genetic recombination or by transformation or by infection since viruses can transfer genes from one organism to another
55
transformation
bacteria/archaea take up raw pieces of dna from environment
56
web of life vs tree of life
web of life=prokaryotes (bc of lateral gene transfer); tree of life=eukaryotes (common ancestor stuff)
57
metagenomics/environmental sequencing
- cataloging all genes present in a complex community of organisms
- typically done with microbial communities
58
unique feature of eukaryotic genes
- wide variation in genome size despite little variation in number of genes
- why? vast amount of repetitive dna (50% of human/mammal genome)
- derived from transposable elements
59
transposable elements
dna segments that can move from one place to another in the genome
60
long interspersed nuclear element (LINE)
a transposable element that codes for reverse transcriptase, when LINEs or other transposable elements move and insert into dna they can cause mutations
- change the structure, function, and evolution of genomes
61
how many LINEs in average genome?
1 million, 20% of your dna; however, most can no longer move on their own in human genome
62
gene duplication
- additional copy of a gene often bc of misalignment of chromosomes during crossing over
- helps to create new genes, evolution contributor
63
gene family
genes that are similar in structure and function, likely shared ancestral sequence
64
unequal crossing over
- can cause gene duplication
- two sister chromatids match up at diff sides when crossing over, gene duplication in one chromatid and gene loss in another
65
duplication and divergence
original gene still functional so if duplicate is mutated a new gene can be added to genome, possibly changing phenotype of organism
66
psuedogenes
- mutation in a duplicated gene copy that is nonfunctional
- about as many pseudogenes as functional genes in human genome
67
human genome project
- took 15 years and $3 bil
- <2% of genome is protein coding
- ~50% is transposable elements
- >25% introns, 17x more abundant than exons
- 95% of human genes are alternatively spliced, about 3+ distinct mRNAs per protein-coding gene
- ~90% of genome transcribed
68
lncRNA (long noncoding rna)
- some important in regulation of gene expression
- do not code for proteins despite being >200 nucleotides long and transcribed
69
functional genomics
how when and where genes are expressed
70
transcriptomics
cataloging rnas and their levels expressed in many genes
71
how many human protein coding genes?
~24k, function of abt 16k known
72
proteomics
large scale study of all proteins in a cell or organism
73
proteome
- how all proteins change over time, vary btwn cells, interact, and lead to phenotypes, complete set of proteins in a cell
- can be determined by mass spectroscopy to determine size of amino acids
74
metabolome
- the complete set of small molecule metabolites present in a biological sample, such as a cell, tissue, or organism
- these metabolites are the products of metabolic processes
75
transcriptome
the complete set of RNA molecules within a cell, tissue, or organism at a specific time and condition
76
dna microarrays
- set of single stranded dna fragments that rep thousands of diff genes are attached to something like a glass slide
- used to determine which genes are expressed in diff cell types, under diff conditions, or at diff dev stages
77
deep-sequencing/rna seq
utilizes next gen sequencing and known whole-genome sequences, compare gene expression btwn diff conditions, more commonly used now than dna microarrays
- reads about 150 bases reliably
78
systems biology
study of structure of network of genes/proteins and how interactions btwn individual network components can lead to emergent biological properties
79
emergent biological properties
- properties that arise at one level of organization from the interaction of elements at a lower organizational level
- ex: phenotypes of a cell emerge from interactions of the molecules that compose it
80
synthetic biology
build biological systems and discover what properties they demonstrate, also engineers living systems for useful purposes
81
dNTPs
deoxynucleotide triphosphates
82
quantitative-reverse transcriptase-pcr (qRT-PCR)
used to test for covid or something
83
high throughput sequencing
these methods are used simultaneously to sequence many diff template molecs
84
ddNTPs
didenucleoside triphosphates (stop reaction in sanger method)
85
massively parallel dna sequencing
thousands to millions of sequencing rxns run at once
86
paralogs
genes that arise via duplication, 2+ paralogs in a genome=gene family
87
how many base pairs does the avg human gene have?
27k
