Test 4 Flashcards
What are plasmids?
Small circular DNA molecules that hold bacterial extrachromosomal genes.
What are the three things that plasmids used in genetic engineering contain?
Antibiotic resistant gene (to select against bacteria that have not taken up the plasmid), an origin of replication, and Polylinkers
What is a polylinker? What is its purpose?
A cluster of unique restriction endonuclease sites. That way there is only one area that plasmid can be cut so that the plasmid is linearized but not cut up into pieces (each one of the restrictions endonuclease sites is unique to this area, and the presence of multiple endonuclease sites means multiple endonuclease can be used.)
What are inserts? What are their purpose?
An insert is a specific DNA sequence (can be gene, ribosome binding site, etc) that is placed into a polylinker to be inserted into bacteria.
Describe the concept of insertional mutagenesis
The creation of mutations in DNA by the addition of one or more base pairs
What type of gene typically makes up a polylinker and allows for selection of bacteria that have a plasmid with the insert in it?
A chromogenic gene, if an insert is placed in a chromogenic gene the gene will not function and the bacteria will no display color
What type of gene is the LacZ gene?
Chromogenic. (Also serves as part of the lac operon)
What are restriction endonucleases?
Naturally occurring enzymes that bacteria use to cut the DNA of invading viruses
Differentiate between endonuclease and exonucleases.
Endonuclease breaks an internal phosphodiester linkage causing fragmentation of the DNA molecule
Exonuclease breaks a terminal phosphodiester linkage in DNA replication (DNA polymerase removing incorrect bases from the strand it is creating)
What are sticky ends? How are they created?
A DNA molecule where each strand ends at a different nucleotide, called sticky because they have a high affinity to hydrogen bond with their complementary pair.
Created by restriction endonucleases.
What kind of symmetry does the recognition sites of sticky ends have? (The recognition site is where they are split apart by the restriction endonuclease)
Dyad symmetry (same 5* -> 3* on both strands)
What creates the linkages between two sticky ends?
DNA ligase forms hydrogen bonds between the sticky ends of two different DNA molecules (hybridization), creating a recombinant DNA molecule
What was the first restriction endonuclease isolated from?
E coli (EcoRI)
What is the issue with using a different species (not the host) promoter in recombinant DNA?
RNA polymerase only recognizes and initiates transcription for promoters of its own species.
Recombinant plasmids must contain the host species promoter before the gene of interest
What major issue can arise from recombining eukaryotic DNA in a prokaryotic host?
The gene of interest from the eukaryote must have it’s introns removed from its coding region
What is the name of the plant pathogen used to genetically engineer plants?
Agrobacterium tumefaciens
How can you insert a piece of DNA into a plant?
A Ti plasmid containing transfer functions (that move T-DNA to the plant from recipient), and T-DNA composed of a plant promoter and coding region are inserted into a plant cell.
What is the issue with producing clotting factor VII?
Codon bias (predisposition to use one codon over another) means that mammalian cells were required instead of E coli
What are the components of CRISPR-Cas9?
dCas9 (similar to a restriction endonuclease), sg RNA 1 and 2 (that tell dCas9 where to cut at each end) and the donor DNA (plant promoter and coding region).
How is CRISPR-Cas9 genetic engineering an improvement upon using a pathogen?
It will cut only in a specific spot that is indicated by the RNA, and it has a higher frequency of inserting DNA.
What is a callus?
the mass of plant cells called after they have been transformed (have DNA inserted) and are growing on agar
What are the next steps to create an adult genetically engineered plant after the plant cells have been transformed?
the cells are grown in two sets of media that have different hormone ratios to promote the growth of both roots and shoots
the plant can then be placed in soil
After the transformed plant has grown up, how do researchers make sure that all of the cells of the plant have the DNA inside?
after the plant is treated with the agrobacterium or crispr and grown into an adult, some of the cells may have not taken up the DNA (mosaic plant), so fully transformed pollen/eggs are obtained so a plant can be made that has DNA in all its cells
Describe the early use of genetic engineering in corn/soybean
Bacillus thuringiensis (BT) toxin was inserted into corn/soybean to protect against lepidopteran larvae (hornworm/cutworm).
BT toxin works by crystallizing in the gut. It is extremely species specific and is used in a powder form as Thuricide
What was the pre-emergent chemical that “RoundUpReady” crops were resistant to?
glyphosate
What type of stem cells are used to create a genetically engineered animal?
embryonic stem cells from a blastocyst
How were blastocyst embryonic cells used to create a genetically engineered animal?
Re-introduce cells into blastocyst → chimeric/mosaic animal → breeding → Embryonic Stem cell derived animal
What is a chimera?
an animal whose cells have been partially genetically engineered
What is SCIDS?
SCIDS (severe combined immunodeficiency syndrome) has defective ADA (adenosine deaminase) that is a part of T-lymphocytes and allows immune response
Why was SCIDS chosen to be the first disease cured by genetic engineering?
Single gene, simple regulation (always on), gene was isolated and cloned, white blood cells could be removed → modified → returned
What was the first method used to treat people with SCIDS?
disarmed retrovirus (RNA as genetic material, host reverse transcriptase converts to DNA integrated into host chromosome)
Disarmed = can’t produce new viruses
Procedure → remove T-lymphocytes → transform with retrovirus → select and amplify transformed cells (selectable marker + induce mitosis with cytokines) → inject into patient
What was the second phase for the treatment of SCIDS?
T-cells do not normally undergo mitosis, so patients had to undergo continuous treatment → need to transform bone marrow stem cells
Same procedure with CRISPR-Cas9 → insert gene into safe harbor (open chromatin = good expression = no genes)
What were the problems with the first method used to treat SCIDS?
2003 2 SCIDS patients got leukemia (retrovirus inserts at random location, may accidentally turn on oncogenes)
How do get from the frequency of alleles to the genotypic frequencies? (#A1, #A2 → A1A2)
2 * (frequency of A1) * (frequency of A2) = frequency of A1A2
Don’t need 2* for homo
How do you get from the genotypic frequencies to the allele frequencies?
Frequency of homozygotes + ½ Frequency of heterozygotes = Allele Frequencies
How do you go from partial genotypic frequencies to allele frequencies? (ex: missing hetero)
If the frequency of one or more is not given, must assume population follows HWE
What is Hardy-Weinberg Equilibrium?
If there are only two alleles for a gene, such that p + q = 1, then the genotypic frequencies will be p2 + 2pq + q2 = 1 and the allele frequencies will stay the same generation after generation
What (5) factors are needed to make alleles into HWE?
(1) random mating, (2) no migration, (3) no mutation, (4) infinite population size, and (5) no selection
If a population is out of HWE, what can/will return it to HWE?
One generation of random mating will put things into HWE (if other factors as well)
What would the HWE equation be for a gene with three alleles?
(a + b + c)2 = a2 + b2 + c2 + 2ab + 2bc + 2ca
What is a polymorphism?
Many forms of a trait/gene in the population
What is DNA fingerprinting?
a way to match DNA samples based on polymorphisms so extreme that every human on the planet has a unique DNA fingerprint
What is a restriction fragment length polymorphism (RFLP)?
The resulting length of a piece of DNA cut with two restriction enzymes
How are different restriction fragment length polymorphisms created (2 main)?
Missing or extra RE site
- a single nucleotide polymorphism (SNP) → one nucleotide changes that creates/destroys a site (3 genotypes for each SNP with 2 alleles)
More or less DNA between RE recognition sites
What is a variable number tandem repeat (VNTP)? Why were they used in DNA fingerprinting?
Sections of DNA with repeating ~15 nu sequences
test for more than one at a time, 100s of VNTRs each with 10-50 alleles
**Example of more or less DNA between RE recognition sites
What were some of the early criticisms of DNA fingerprinting and how were they addressed?
Not all alleles are of equal frequency (may have 30 alleles, but 2 account for 99%)
More sampling to more accurately describe allele frequencies
Assume independence among alleles at different VNTRs
More sampling to determine linkage equilibrium (alleles turned out to be independent)
What are short tandem repeats (STRs)?
like VNTRs, but with fewer bases in each repeated section → many more alleles and more sites (called satellites)
How did the invention of polymerase chain reactions (PCRs) change DNA forensics?
More reliable results with less DNA and fewer steps
What is a target sequence in PCR?
Known sequence of DNA that contains the short tandum repeat or other region that you want amplified
What components are needed for PCR?
2 primers (one for each strand of DNA), template DNA, taq polymerase (a temp-resistant DNA polymerase), and free nucleotides
How does PCR work?
Cycle 1: Raise temp to break apart strands of DNA → two primers (one for each strand) that bind to the ends of the target sequence → synthesis temperature + taq polymerase + free nucleotides → synthesize two pieces of DNA that include the target sequence and extend beyond it to one side (taq polymerase does not know where to stop
Cycle 2: Raise temp to break apart strands of DNA → excess primer → synthesis temp and taq polymerase → 2 template strands, 4 with excess DNA on both sides, 2 with excess DNA on only one side
Cycle 3: Produces 6 larger molecules and 2 molecules that are the length of the target sequence
Cycle 4: Produces 8 larger molecules and 8 that are the right length
Repeat for 30 cycles to get 60 long molecules and 1,073,741,766 the length of the target sequence
How are more alleles created for STRs and VNTRs?
Unequal crossover at meiosis