3.1.2 DNA Manipulation Flashcards
(59 cards)
1
Q
restriction endonuclease
A
enzymes that cut strands of DNA at specific recognition sites on the phosphate sugar backbone
2
Q
recognition site
A
4-6 nucleotides specific to each enzyme usually palindromes read from 5’-3’
specific sequence of DNA that a particular restriction enzyme cuts at due to its active site being complementary to this recognition site
3
Q
sticky
A
- don’t cut in middle of recognition site meaning overhanging unpaired nucleotides that want to stick to complementary nucleotides are created
- useful for inserting DNA in correct orientation
4
Q
blunt
A
- cut in middle of recognition site
- useful for separating DNA
5
Q
ligase
A
joins two fragments of DNA together by catalysing formation of phosphodiester bonds between fragments
6
Q
polymerase
A
- joins monomers together to form polymer
- adds nucleotides to DNA or RNA leading to copying entire genes
7
Q
primer
A
short single stranded chain of nucleotides complementary to template strand attaches to DNA template so polymerase can read and synthesise complementary strand in 5’-3’ direction
8
Q
polymerase chain reaction (PCR)
A
- amplifies DNA by creating multiple identical copies (doubled each time)
- condensation polymerisation reaction where nucleotide monomers are linked together in a chain to form DNA polymer and releasing water molecules
9
Q
PCR ingredients
A
- DNA sample
- primers
- Taq polymerase
- nucleotide bases
- buffer (carry current and maintain ph)
10
Q
PCR process
A
- denature: DNA heated to 90-95 degrees to break hydrogen bonds between bases and separate strands to from single stranded DNA
- anneal: single stranded DNA cooled to 50-55 degrees to allow primers to bind to complementary sequences on 5’ end of both single stranded DNA
- extend: DNA heated to 72 degrees to allow Taq polymerase to work optimally and bind to primer to begin synthesizing new complementary strand of DNA by adding free nucleotides to 3’ end
- repeat
11
Q
gel electrophoresis
A
- sorts DNA fragments according to size
- larger fragments move slower and less far due to more friction
12
Q
gel electrophoresis components
A
- gel made from agarose
- DNA fragments stained with fluorescent dye
- samples of DNA fragments loaded into wells at negative end
- DNA is negatively charged so when electricity applied, moves to positive end
13
Q
molecular weight ruler
A
- contains fragments of DNA of known base pair length that are used to compare DNA samples and estimate size
- needed due to voltage, gel composition, buffer concentration and time affecting distance DNA travels
14
Q
probes
A
- radioactively or fluorescently labelled single strand of nucleic acid (DNA or RNA) complementary to known target DNA sequence that hybridises with it and isused to identify this target sequence in gel electrophoresis
15
Q
genetic testing
A
- examine person’s DNA to detect genetic diseases
- PCR, restriction enzyme, gel electro
- make decisions about having children, early intervention
16
Q
DNA profiling
A
- compare individuals’ DNA to establish their identity using unique sequence of nucleotides in their DNA
- PCR of STR or RFLP, gel electro
17
Q
naming restriction enzymes
A
- species of bacteria isolated from (Eco)
- strain of bacteria (R)
- number stating order which it was discovered in that strain (1)
18
Q
genetic screening implications
A
can affect individual or gene pool as alleles can be selected against and not passed onto next generation
19
Q
restriction fragment length polymorphism (RFLP)
A
- highly variable (polymorphic) region amplified by PCR and cut using restriction enzymes to separate by gel electro
- DNA from different people has different number and position of recognition site resulting in unique number of different sized fragments
20
Q
short tandem repeats (STR)
A
- short repeated nucleotides of varying length found in introns of autosomal chromosomes
- not affected by natural selection resulting in hundreds of variants
- different between two alleles of one person and different between people
- same flanking regions so same primer can be used to initiate PCR
21
Q
plasmid
A
small circular loops of bacterial DNA separate that replicate independently of chromosomes and act as vectors to transport foreign DNA into bacteria
22
Q
recombinant plasmid
A
plasmid edited to integrate target gene
23
Q
bacterial transformation
A
bacteria take up foreign DNA from environment
24
Q
reverse transcriptase
A
- transcribes mRNA backwards to make cDNA with no introns so bacteria can use it
25
plasmid vector contains
- restriction endonuclease recognition site
- antibiotic resistance genes
- reporter gene with easily identifiable phenotype
26
DNA ligase
-primase adds RNA primer
- DNA polymerase extends from the RNA primer backwards
- creates okazaki fragments with gaps between them
- DNA polymerase replaces RNA primer with DNA nucleotides
- DNA ligase joins fragments
27
GMO bacteria transformation
1. mRNA coding for protein is isolated from human cell
2. reverse transcriptase copies mRNA to make cDNA
3. plasmid vector isolated from bacteria
4. human gene of interest (cDNA) and plasmid cut with same restriction enzyme producing complementary sticky ends
5. plasmid and cDNA hybridize and DNA ligase bonds sugar phosphate backbone
6. recombinant plasmid acts as vector to transport gene into bacteria
7. transfomed bacteria detected by agar plate with antibiotics
8. bacteria with recombinant plasmid detected by selectable trait from reporter gene
9. cloned to make human protein
28
GMO plant transformation
- plasmid is Ti plasmid
- agrobacterium (bacteria with special plasmid that transports foreign DNA into linear DNA of plants)
- herbicide resistance
29
insulin
- quaternary structure with two polypeptide chains, (alpha and beta subunits)
- requires two different recombinant plasmids and two different transformed bacteria samples to produce each subunit since bacteria can't cleave the two chains then join them to form quaternary structure since they lack membrane bound organelles
- joined by disulphide bridge
30
making human insulin
bacteria:
- genes for chain a and b inserted into different plasmids next to lacZ and strong promoter (RNA polymerase to bind and transcribe insulin often)
- each plasmid used to transport different E. coli
- transformed E. coli make fusion proteins of beta-galactosidase with a or b chain (so bacteria can't break it down)
- fusion protein chemically treated to remove beta- galactosidase
- chain a and b joined to form insulin
yeast:
- genes for chain a and b added to large plasmid in yeast
- eukaryotic yeast cells modify and combine a and b chains
31
genetic engineering
altering organisms' genome using genetic recombination technologies
- genes can be silenced, inserted, removed, altered
32
GMO
DNA manipulated by humans in lab
33
TGO
DNA from different species inserted meaning they can produce proteins not previously in their proteome (speciation occurs)
34
gene therapy
- replaces faulty genes with functional genes to try and eliminate disease caused by mutant allele
- by direct microinjection or viral/liposome delivery
35
agricultural uses of gene therapy
- increasing crop yield
- increasing nutritional value
- ability to grow in different conditions
- resists diseases and other environmental factors like droughts
36
biological pros
- less land due to better productivity
- insect resistant GMOs require fewer pesticides
- improved nutritional content improves human health
37
biological cons
- lose effectiveness if weeds or pests evolve resistance
- loss of genetic diversity within population
- cross pollination between GM crops and wild species leading to genes spreading and unforeseen circumstances
38
social pros
- better food security
- ability to grow in adverse conditions prevents famine
- labour demands since pesticides don't need to be sprayed and weeds don't need to be pulled out by hand
- larger profit for farmer
- improved flavour and texture
- healthier human population
39
social cons
- buying new seeds each season is costly
- complex legal issues cause farmers stress
- strict packaging and marketing regulations for GMO producers may not be complied with if producer or consumer uneducated
40
ethical pros
- ethical imperative to help improve nutrition, wealth, health especially in developing nations
41
ethical cons
- unnatural, playing god
- unsafe to eat
- modifying animals for human benefit inhumane
- companies owning rights to GM crops and making unfair demands to farmers
- cross pollution of non-GM crops to nearby GM crops making farmers sued
- can't reuses seeds, must be bought new each year
42
CRISPR Cas 9
enzyme and guide RNA that edit DNA by cutting it at sequence complementary to guide RNA
43
CRISPR Cas 9 in bacteria
exposure: - bacteriophage injects DNA into bacteria which identifies it as foreign
- Cas1 and Cas2 enzymes look for PAM sequence and cut out short section of viral DNA (protospacer)
- inserted into CRISPR array on 5' end
expression: - pre-CRISPR RNA is transcribed
- tracer RNA anneals to repeat regions
- RNAase cuts repeat region creating crisper-tracer RNA
extermination: - guide RNA and Cas9 combine to become CRISPR Cas9 complex
- scans to cell looking for PAM site (ensuring not its own DNA) and complementary viral DNA and binds with this DNA
- Cas9 cleaves phosphate sugar backbone and two active sites to cut both strands and produce blunt ends
- enzymes in bacteria naturally act to repair cut DNA but mostly fail resulting in mutations that render DNA non-functional
- if no mutation occurs, process repeats until DNA repair fails
44
Clustered Regularly Interspaced Short Palindromic Repeats
- short and palindromic repeated sequences
- interrupted by spacer DNA
- downstream of Cas9
45
CRISPR Cas9 in gene editing
- single guide RNA which is created in a lab and single stranded is combined with Cas9
- sgRNA and Cas9 mixture injected into specific cell
- Cas9 finds target PAM sequence adjacent to complementary DNA to sgRNA and binds to PAM
- Cas9 cuts DNA sequence and cell attempts to repair it, this is where scientists can add new nucleotides, replace nucleotides or silence the gene
46
CRISPR applications
- locating genes by adding fluorescent gene
- gene knockout: silencing expression of genes to identify its function
- replace deleterious alleles with healthy ones
- decrease susceptibility to diseases
47
CRISPR limitations
- successful in animals not humans yet
- new genes may not be taken in
48
non maleficence for
reduce disadvantage or pain faced by child and parents of children with genetic conditions
49
non maleficence against
possibility of off target cleavages and mosaics( cells with edited and non edited genomes) posing safety issues
50
respect for
alleviate suffering caused by genetic disorders preserving dignity of individuals who would otherwise live with disease
51
respect against
cannot get informed consent from embryos to edit genes
dismiss value of people with disease
52
justice for
give everyone a fair chance to live a full life
53
justice against
only wealthy people are able to treat genetic conditions
threat to those who are judged by society as biologically inferior
54
integrity against
informed consent and transparency is required
genetic information could be used for other purposes
55
beneficence for
eliminates disease and suffering
56
DNA polymerase
enzyme that links nucleotides by phosphodiester bonds to synthesise DNA
57
vector
transports gene into new host to make it a GMO
58
selectable marker
codes for protein allowing scientists to select genetically engineered cells by their ability to survive under certain conditions
59
offtarget edit
mismatch between base pairs allowing sgRNA to bind to nontarget DNA
