Ch. 17 Flashcards
(32 cards)
1
Q
Recombinant DNA
A
A single DNA molecule made from two different sources. Ability to isolate and manipulate DNA revolutionized biotechnology
2
Q
Restriction endonucleases
A
Enzymes that cleave DNA at specific sites
- used by bacteria against viruses
Discovery of restriction endonucleases important because
- ability to cut DNA into specific fragments
- use in genome mapping
3
Q
3 types of restriction enzymes
A
- type I and III cleave with less precision and are not used in manipulating DNA
- type II
• recognize specific DNA sequences; most sites are palindromes
• cleave at specific sites within sequence
• can lead to “sticky ends” that can be joined. Blunt ends can also be joined
4
Q
Gel electrophoresis
A
- separate DNA fragments by size
- larger fragments move slower, smaller move faster
5
Q
DNA ligase
A
- joins the two fragments forming a stable DNA molecule
- catalysts formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides
- same enzyme joins Okazaki fragments in lagging strand in replication
6
Q
Reverse transcriptase
A
- can use RNA as a template to make DNA
- DNA made from copying mRNA is called complementary DNA (cDNA)
• allows analysis ofbonly sequences that are actually used to synthesize proteins
7
Q
DNA libraries
A
- a collection of DNA molecules that can be maintained and replicated in a host organism
- DNA of interest inserted into cloning vectors- plasmids or artificial chromosomes
8
Q
DNA libraries: cloning vectors contain
A
- a sequence that allows replication in a host organism
- a selectable marker
- sequences that allow DNA fragments to be added
9
Q
cDNA libraries
A
- cells from a specific time point or tissue can be used to isolate mRNA for cDNA synthesis
- cDNAs used to construct a library that represents only the genes expressed at that time point or in that tissue
- all genomic libraries from a cell will be the same but cDNA libraries can be different
10
Q
Polymerase Chain Reaction (PCR)
A
- developed in 1993
- mimics DNA replication to produce millions of copies of a DNA sequence
- allows the amplification of a small DNA fragment using primera that flank the region
11
Q
Each PCR cycle involves three steps
A
- denaturation (high temperatures)
- annealing of primers (low temperature)
- DNA synthesis (intermediate temperature)
• taq polymerase
12
Q
Reverse transcription PCR
A
- PCR is performed on cDNA made from mRNA
- called reverse transcription PCR (RT-PCR)
- allows creation of recombinant DNA containing only the exons of genes
13
Q
Quantitative RT-PCR
A
- involves isolating mRNA converting to cDNA using RT, then using PCR to amplify specific cDNAs
- amount of DNA produced can be measured in real time by the PCR machine
- can be quantitated using DNA-binding dyes or DNA-binding probes
14
Q
PCR in sequencing
A
- preciously DNA has to be cloned into a vector to be sequences; laborious and costly
- “next generation” sequencing use PCR techniques for sequencing
- sequencing is now quick and inexpensive
15
Q
DNA fingerprinting
A
- need to identify an individual based on a small amount of tissue or body fluids
- takes advantage of short tandem repeats (STRs) that vary among individuals. Population is polymorphic for these markers
- PCR primers flank a region known to contain an STR
- using several probes, probability of identity can be calculated or identity can be ruled out
16
Q
Creating point mutations using PCR
A
- geneticists previously creates mutations randomly using chemicals
- specific mutations can now be introduced using PCR primers with a slightly different sequence than the template
- can also use PCR to create random mutations using specific conditions
- effects of introduced mutations can be analyzed in various ways
17
Q
RNA interference
A
- in some experimental systems it is not possible to make and analyze mutants in a living cell
- allows researchers to reduce the amount of a gene product in living cells/organisms
• degrades or blocks translation of a specific mRNA in cells or organisms
• requires production of a short double-stranded RNA complementary to the mRNA that encodes protein of interest
18
Q
Direct editing of the genome
A
- until very recently it has not been possible to alter the DNA in a living cell
- discovery of several proteins means it is now possible to quickly and easily change a genes sequence directly in a cell
- two main ways to edit genes in vivo
• tale (transcription activator-like effector) proteins
• CRISPR
19
Q
TALE proteins
A
- different TALE repeat domains bind to different nucleotides
- by combining different TALE repeat domains, different sequences of DNA can be bound by TALE proteins
- a TALE-nuclease fusión (TALEN) can be used to cleave a specific DNA sequence in vivo; genes can be inactivated
20
Q
Transgenic organisms
A
- transgene= gene from another species
- genetically modified organisms have been genetically altered by techniques other than conventional breeding
- universal nature of genetic code makes it possible to put a human gene in E. coli, etc.
- removal or addition of a gene can help scientists understand the genes function
21
Q
“Knockout” mice
A
- clones gene interrupted by replacement with a marker gene
- marker gene codes for resistance to the antibiotic neomycin
- interrupted gene is introduced into embryonic stem cells (ES cells)
- ES cells injected into embryo early in development
22
Q
“Knockin” mice
A
- have a normal allele replaced with allele that has a specific genetic alteration
- can introduce alleles that result in complete loss of function, partial loss of function, or gain of function
23
Q
Genetic modification of plants
A
- unlike animals, it is generally not possibly to target a specific gene sequence in plants; integration of transgenes into plants is random
- DNA introduced into plants by electroporation, physical bombardment, chemical treatment, bacterial transfer
24
Q
Ti (tumor-inducing) plasmid
A
- commonly used vector for plant genetic engineering
- obtained from agrobacterium tunefaciens
- gene of interest can be engineered into the Ti plasmid
- part of the Ti plasmid (including the gene of interest) integrates into the plant DNA
- can use to grow a mature plant in which all cells contain the transgene
25
Medically important proteins can be produced in bacteria
Like human insulin.
26
Fluorescent in situ hybridization (FISH)
- FISH uses hybridization to detect a specific DNA in a complex mixture
- labeled DNA probe can find target DNA within chromosomes
- detects large deletions, inversions, duplications, and translocations
- useful in characterizing cancer
• HER2 duplication in breast cancer
• allows appropriate drugs to be administered
27
DNA microarrays (gene chips)
- collections of hundreds to thousands of different DNA sequences spotted onto a solid surface
- can look for biomarkers associates with disease; can look at mRNA to tell which genes are being expressed in certain conditions
- can be used to diagnose genetic disorders
28
Environmental biotechnology
- biofuels are fuels derived from biomass made from recently fixed carbon sources (usually crop plants or algae)
- renewable; more environmentally friendly than fossil fuels
- microalgae cultures in large open ponds or in self-contained culture systems called photobioreactors
29
Treatment of hydrocarbons in environment
- bioremediation= degradation or metabolism of hydrocarbon pollutants by microorganisms
- bioremediation of hydrocarbons important for breakdown of hydrocarbon-derived fuels in the ecosystem
- potential to genetically engineer microorganisms to better break down hydrocarbons
30
Agricultural applications of biotechnology
Herbicide resistance
- broadleaf plants have been engineered to be resistant to the herbicide glyphosate
Benefits:
- crop resistant to glyphosate would not have to be weeded
- single herbicide instead of many types
- glyphosate breaks down in environment
In the US, 90% of soy currently grown is GM soy
31
Agricultural applications of biotechnology (2)
Bt crops
- insecticidal proteins have been transferred into crop plants to make them pest-resistant
- bt toxin from Bacillus thuringiensis
- use of bt maize is the second nose common GM crop globally
Stacked crops
- both glyphosate-resistant and bt-producing
32
Social issues with transgenic crops
Adoption of GM crops has been resisted in some areas because of questions
- crop safety for human consumption
• no documentation of adverse effect on human health
- movement of genes into wild relatives
- possible loss of biodiversity
