Ch. 10 Biotechnology Flashcards
(90 cards)
1
Q
What is Genetic engineering?
A
moving genes from one organism to another
2
Q
What is Genetic modification?
A
DNA alterations caused by mutagens
3
Q
What is Biotechnology?
A
using microbes, such as yeast to produce alcohol
4
Q
What is GMOs?
A
genetically modified organisms, such as plants, animals,or microorganisms
5
Q
Is Selective breeding genetic engineering?
A
No
6
Q
What ingredients for clone gene?
A
Gene of interst, restriction endonuclease enzyme, vector, Ligase, Host cell
7
Q
What does restriction endonuclease enzyme do?
A
It cuts DNA at specific sequences
8
Q
What kind of vector is needed for clone gene?
A
vector that carries the inserted gene; most commonly bacterial plasmid, but also other types of vectors
9
Q
What must vectors able to do?
A
replicate so that inserted gene is copied and be transcribed and translated to produce functional protein
10
Q
What is ligase?
A
enzyme used to bind DNA together
11
Q
What host cell is to be used for clone gene?
A
usually bacteria but could also be yeast or animal cells Takes up recombinant plasmid.
Will replicate and copy the DNA as well as transcribe and translate inserted gene
12
Q
What are Restriction endonucleases?
A
special enzymes that bind to specific short sequences of DNA and make cut
13
Q
How long these short sequences?
A
These short sequences are usually four to six nucleotides long
14
Q
What is structure of these short sequences?
A
These sequences are symmetrical in that DNA double helix has identical sequence running in opposite directions: referred to as palindromes
15
Q
How rare is cut made by restriction enzymes?
A
Cut made by restriction enzymes is also unusual
16
Q
Where is cut made?
A
cut is not necessarily made in centre of sequence, but to one side
17
Q
What does cut to one side cause?
A
This creates break with short, single strands of DNA dangling from each end
18
Q
What are sticky ends?
A
Overhangs caused by cut in side
19
Q
What creates cuts with same restriction enzymes?
A
Same sticky ends
20
Q
Where do restriction enzymes come from?
A
Bacteria
21
Q
What can restriction enzymes be used?
A
to fight viral infections, and they can also select for gene sequences in strands that they take in through transformation
22
Q
How many of different restrction have been discovered?
A
Hundreds of them
23
Q
What are some examples of restriction enzymes?
A
EcoRI 5’ GAATTC 3’
BamHI 5’ GGATCC 3’
HaeIII 5’ GGCC 3’
24
Q
What do sticky ends make possible for?
A
recombination of fragments generated by cut restriction enzymes
25
What can fragments with same sticky ends do?
Fragments with same sticky ends can pair up and be resealed by DNA ligase
26
Where can these DNA fragments can be from?
they can be from different species
27
What should all vectors must have in order for them to be useful to genetic scienctists?
Certain charateristics
28
List certain chracteristics that vectors should have.
1.They must be able to replicate within host cell
2.They must contain restriction enzyme cut sites
3.They should carry selectable marker to allow for identification of host cells that contain recombinant DNA
4.They must have promoter region for expression of inserted gene
5.vector should be easy to recover from host cells that contain it
29
What is transformation?
Process that allow Bacteria become easy to grow and will easily take up plasmids
30
What is selectable marker?
antibiotic resistance gene that many plasmids have been engineered to contain origin of replication
31
Where are many restriction enzyme usually placed?
They are placed into multiple cloning site (MCS) that is sometimes found within another selectable marker, such as lacZ gene
32
Why do vectors use bacteria as host?
Because they are easy to grow, replicate gene when they replicate their own DNA, before they divide by binary fission, and reproduce themselves by millions overnight; therefore, many copies of gene can be made in one day
33
What are two ways to identify cells carrying this plasmid?
1. Cells are able to grow on medium containing ampicillin (ampicillin resistant)
2.Cells will be blue when grown on medium containing X-gal if lacZ is intact
34
What does lacZ breakdown?
They break down lactose Into glucose and galactose; also breaks down Lactose analogue called X-gal
35
What does breakdown of X-gal produce?
product that turns bacterial cells blue
36
What would happen if piece of DNA is inserted into MCS (multiple cloning site)?
non-functional LacZ enzyme is made, cells remain white since X-gal is not broken down into blue forming product
37
What does it mean there is blue product by breakdown x gal from blue colonies?
That they do not cotain genes meaning they are not recombinant DNA
38
What does white product by breakdown x gal from white colonies mean?
That they do have genes meaning they are recombinant DNA
39
What are bacteriophages?
Viruses that infect bacteria
40
What can all of lambda phage can be used to?
to carry genes to bacterium
41
What can genes involved in lysogenic phase be?
They can be removed and replaced with foreign DNA without affecting ability of phage to infect bacteria and replicate
42
What is transduction?
Process of phage-containing recombinant DNA can then infect bacterial cells
43
What are cosmids?
laboratory-constructed vectors that contain cos site from lambda phage, which is required for packaging chromosomes into virus particles
44
What do cosmids contain?
plasmid features such as antibiotic resistance and origin of replication
45
How much size of sequences of DNA do cosmids carry?
They are used to carry much larger sequences of DNA than plasmids
46
Why are Yeast artificial chromosomes (YACs) beneficial?
because these vectors can be grown in yeast cells, which are eukaryotic
47
Why is eukaryotic particulary beneficial?
Because prokaryotes lack ability to process RNA
48
What does origin of replication for YACs do?
centromere allows for DNA can be distributed during mitosis, restriction enzyme cut sites, and yeast-specific selectable markers
49
What are the chracteristics of yeast cells?
Yeast cells are easily grown and have been used to express many mammalian genes
50
What is process of cloning gene in numerical orders?
1. Extract and purify DNA (from human and bacteria).
2. Digest human DNA with restriction enzymes.
3. Cut plasmid (bacterial DNA) with same restriction enzyme.
4. Insert DNA into plasmid to make recombinant plasmid
* Which enzyme “glues” DNA together?
* Ligase
5. Make bacteria take up DNA (transformation).
* Recall three ways bacteria take up new DNA
6. Grow bacteria overnight to make millions of copies.
7. Spread bacteria on culture dish to separate individual clones to
work with gene.
51
What can genetic programs can be used to?
used to obtain DNA sequences of many genes and restriction enzyme cut sites that are found within genes and in sequences surrounding them (Restriction Map)
52
What does knowing genetic programs allow for?
That appropriate restriction enzymes can be chosen to cut DNA to obtain whole gene (part of gene is not very useful for most genetic engineering applications)
53
What is screening process?
First, find clones that have taken up vector; if bacteria did not take up vector, it will NOT be resistant to ampicillin and they will not grow.
Second, find clones that did take up vector AND have human DNA inserted into multiple cloning sites
54
What is one of genes that code for protein that breaks down lactose
LacZ
55
What would happen if using substrate similar to lactose called X-gal?
bacteria will turn blue as they digest it
56
What would happen if gene is inserted into MCS
lacZ gene will be disrupted and bacteria will NOT turn blue; they will be white meaning only white colonies will have recombinant DNA
57
What is recombinant somatotropin (type of growth hormone) used to do?
It is used to increase milk production as they genetically engineer hormone
58
Is recombinant somatotropin legal in Cadana?
No
59
What can DNA fragments be before DNA inserted into plasmid?
DNA fragments can be sorted based on their size
60
What is Gel electrophoresis used to?
used to separate strands of DNA by electrical current
61
What charge is DNA?
DNA has negative charge (remember this is why we need histones to condense DNA) and will move toward the + end
62
Is it bigger or smaller fragments that run faster through gel?
Smaller fragments run faster through gel
63
What is number of base pairs that will not be whole gene?
Anything smaller than 1000 base pairs
64
What is Polymerase chain reaction (PCR)?
technique to generate multiple copies of DNA
65
What part of DNA are first synthesized?
Short sequences of DNA called primers are first synthesized
66
Where do primer sequences occur?
primer sequences occur on either side of DNA region to be amplified—5 primer and 3 primer are needed
67
What is PCR technique compared to bacteria?
PCR technique is way to generate lot of DNA of interest quickly rather than rely on bacteria to produce copies
68
What are 3 steps involved in PCR
1. Denaturation which separates DNA strands
2. Primer annealing where 5' and 3' primers have to bind to single-stranded DNA
3. Primer extension which DNA polymerase replicates DNA in between two primers
69
What ingredients does PCR need?
1.Template DNA, such as plasmid with insulin gene
2. 3' and 5' primers, which were made specifically for insulin gene
3.Nucleotides (A, C, T, and G)
4.Polymerase (Taq polymerase is not denatured in high heat as it is from bacteria
that live in geothermal hot springs)
70
Where are ingredients mixed for PCR?
Ingredients are mixed in microfuge tube that will fit into PCR machine
71
What is happening in PCR cycle?
95 degrees for 2 min to denature DNA,
55 degrees for 1 min to anneal primers to template DNA,
75 degrees for 1 min for every 1 kb—optimal temperature for polymerase to add nucleotides
72
How many times cycles are repeated?
approximately 30 times
73
What is RT-PCR?
Process that uses enzyme reverse transcriptase to convert mRNA molecule into cDNA molecule meaning No Introns
74
What is RT-PCR good for?
Good for inserting eukaryotic DNA into bacteria to amplify
75
What can RT-PCR also mean?
RT-PCR can also mean "real time" PCR (quantitative PCR, or qPCR), which is process that uses dye or fluorescence that binds to DNA, which can then be used to quantify amount of DNA being amplified at various cycle times
76
What is DNA finger printing?
That everyone has varying lengths of repeated DNA sequences in their introns
77
What is variable number tandem repeats (VNTRs)?
repeated sequences are of different lengths in each person, like finger print
78
How will VNTR be used in crime scene in order of steps?
1.DNA is digested with restriction enzymes.
2.DNA fragments are run on gel to separate varying sizes of DNA.
3.DNA is then transferred to a piece of filter paper
4.transferred DNA is denatured into single strands.
5.A probe (radioactively labelled) that will label VNTRs is hybridized to DNA on filter paper.
6.DNA from crime scene is compared to suspect’s, or compare family members, such as paternity testing
79
What is gene therapy?
Process of inserting gene into an individual’s cells to replace mutated gene
80
What is goal and difficulty of gene therapy?
Goal is to restore cell’s normal function with healthy gene, difficulty is that inserting particular gene into correct location in genome in correct cell type
81
What is Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)?
CRISPR is new technology that involves family of DNA sequences found in bacteria and are derived from fragments of bacteriophages
82
What is Cas9?
restriction enzyme that recognizes palindrome sequences and it also contains specific guide
83
What can genetically engineered animals do?
Since these animals are eukaryotic, they can process RNA and modify proteins in Golgi
84
What is BIoSteel?
spider silk in their(goats') milk
85
What is silk gene isolated from spiders attached to?
attached to promoter involved in regulating milk production genes in goats
86
What is DNA microinjected to in BioSteel?
nucleus of egg of animal
87
Where is BioSteel used?
Purified and spun into fibres, these fibres of BioSteel are used to make bulletproof vests
88
For what purposes did genes in crop plants have been successfully manipulated through genetic engineering
1. To make plants more resistant to insects - e.g., Bt corn
2. To make plants resistant to herbicides – e.g., glyphosate resistant
3. To prevent ripening - e.g., flavr savr tomatoes (although not as
common any more due to cost of production)
4. To make plants hardier against environmental stress - e.g., cold-
tolerant
5. To make plants produce antiviral chemicals - e.g., papaya trees
6. To add nutrients, - e.g. beta carotene in Golden rice
89
What potential problem does engineering crops have?
They have Bacillus thuringiensis (Bt) which is soil bacterium that produces protein
that is toxic when eaten by crop pests and it is said to be harmless but sounding like selection pressure
90
What are most commonly expressed concerns about GMO foods?
1.possible creation of allergens
2.pesticides possibly being expressed in parts of plant that are
consumed
3.toxic load of herbicides sprayed on herbicide resistant crops
4.possible reduction of micronutrients in GMO foods if they are grown in
poor/ depleted soil
5.It is very difficult to conduct long term studies that could clearly identify
any health issues as being specifically linked to GMOs foods
6.Research continues use conclusions statements such as "no overt consequences", or "not likely to present risks to humans"
