Exam 3-my Q's Flashcards
What is a mutation?
a heritable change in a DNA sequence
what are the classifications of genes by what chromosome the gene is location on?
autosomal, x linked, somatic, germ line (germinal)
are somatic mutations inherited?
no, they are not passed down
are germ line mutations inherited?
yes, if the mutation affects germ cells of progeny
What are the classifications of mutations by type of molecular change
nucleotide substitution
insertions and deletions
What are the types of nucleotide substitutions, define them
transition: pyrimidine replaced with other pyrimidine, or purine with purine
transversion: pyrimidine replaced with purine or vice versa
how are transversions and transitions further classified? define
silent- changes codon into another that codes for the same AA
missense- results in AA change
nonsense- changes a codon into a stop codon
what are the 2 types of missense mutations
conservative- chemically similar AA
non conservative- chemically different AA
What kind of mutation causes sickle cell anemia
non conservative missense mutation, nucleotide substitution
what is a frameshift
if there is an insertion or deletion of any # of nucleotide pairs that is not a multiple of 3, the reading frame changes
What is a frameshift likely to cause?
usually truncated non functional proteins
what is a nonsense mutation likely to cause
truncated pp, might still be functional
What are the mutation classifications by function/ phenotype (9)
neutral- no effect
null- complete function loss
leaky- reduced function
loss of function-decreases or eliminates function
gain of function-
lethal-
nutritional- affects biochem pathway
conditional- only appears under certain factors (ex temp)
suppressor mutation- 2nd mutation that reverses the original
are loss of function mutations often recessive or dominant?
recessive
are gain of function mutations often dominant or recessive?
dominant
How does UV light affect DNA?
it causes pyrimidine dimers that are bulky, DNA polymerase cant replicate it and skips it. can lead to a mutation
How do DNA polymerases deal with UV damage in DNA
some can replicate over the dimer by replacing the pyrimidine dimers with A’s by default.
Describe the process of mismatch repair
happens after DNA replication if anything is missed.
incorrect base is cut
the parent strand is identified my methylation
a part is excised and replaced
DNA ligase joins gaps
What is the mutation rate after mismatch repair
1x10^9 nucleotides
explain how DNA is repaired by photoreactivation repair
UV damage is reversed, PRE photolyase cleaves the bond between T’s only in the presence of light.
What kind of organisms can perform photoreactivation repair
bacteria and yeast, not higher eukaryotes
Explain base excision repair
damaged non bulky chemically modified or inappropriate bases can be removed this way
- An incorrect base is excised and an AP site is left in its place (empty spot)
- AP endonuclease cleaves the AP site and the nucleotide is removed
- a nucleotide is added with the other strand as a template
- DNA ligase covalently joins the nucleotides.
explain nucleotide excision repair
can be used to remove bulky pyrimidine dimers and stretches out damaged DNA.
- the damaged strand is cut upstream and downstream of the damaged site.
- new DNA is synthesized using undamaged strand as template
- DNA ligase fills gaps
If the base pair AT experiences a transition mutation, what would the result be?
AT is replaced with GC
What can mistakes in DNA replication lead to? how does it happen?
insertions or deletions.
one strand can slip, creates a looped out nucleotide.
What strand slippage leads to insertions? deletions?
insertion- if new strand slips
deletion- if template strand slips
during replication
What are spontaneous lesions?
naturally occurring DNA damage
caused by depurination, could lead to random ntd replacement.
What does the deamination of a cytosine lead to?
uracil
what are mutagens what can they do
they cause DNA damage and can act by replacing, altering or damaging a base
they are an agent (chemical or radiation) that causes an increase in the rate of mutation
What are the 4 types of mutagens
base analogs, alkalating and intercalating agents, UV light and ionizing radiation
explain what alkylating agents and intercalating agents do
alkylating- transfer alkyl groups to DNA converting nitrogenous bases to unwanted chemicals
intercalating- create a wedge between base pairs
explain what ionizing radiation does
x rays result in oxidative DNA damage that can lead to mutations
what do restriction endonuclease enzymes do and how are they used
cleave phosphodiester bonds between nucleotides within a nucleic acid, they are used in recombinant DNA technology
where are restriction endonucleases seen naturally and what is their function
in bacteria, function is to protect bacteria from infection of bacteriophages
how do restriction endonucleases know what to do?
they recognize and bind to restriction sites in DNA and cleave at a specific location in that site.
explain what a staggered cut is
REs make staggered cuts when DNA fragments have hanging ends that are sticky
explain what a blunt end is
REs cut right in the middle of the restriction site where DNA strands do not have sticky ends
what are DNA cloning vectors
DNA molecules that accept DNA fragments and replicate them in a host cell
What are the requirements for a cloning vector
- several common restriction sites
- must be able to be introduced into a host cell
- must have the ability to replicate independently or have a oric
- must have a selectable marker (like an antibiotic resistant gene)
- may have specific sequences that allow for sequencing inserted DNA
- may have a gene that identifies host cells that carry recombinant plasmids
List the steps to cloning a DNA fragment
- plasmid is removed from bacterial cells and cut with an RE vector, the DNA of interest is isolated and cut with the same RE in order to create compatible sticky ends
- DNA fragments are added together along with DNA ligase to form a recombinant DNA
- Ligation mixture is introduced into host cells by transformation
- transformants (bacterial cells that took up the plasmid) are identified by growing on selective medium
Explain blue to white screening
allows for the identification and selection of recombinant plasmids.
when lac z and beta galactosidase cleave to x gal a blue color is created
Explain blue to white screening
allows for the identification and selection of recombinant plasmids.
when lac z and beta galactosidase cleave to x gal a blue color is created
what is needed for a blue to white screening to work
intact lac z gene and at least 1 restriction site
what are the possible outcomes of the blue to white screening test
- if insert is not ligated into lac z gene, the multiple cloning site will H bond back together resulting in blue colonies
- if insert is ligated into the lac z gene, the reading frame of the plasmid is interrupted producing a different protein other than beta galactosidase, results in white colonies
What are the possible methods of testing if your plasmid took the clone gene
PCR
sequence the plasmid
white to blue screening
What are DNA libraries and how are they constructed
constructed from many overlapping fragments of the genome.
the genomic DNA (gDNA) is extracted from cells and cut randomly with REs to break into fragments
the overlapping fragments are inserted individually into cloning vectors
it is a collection of recombinant DNA containing bacteria representing the entire genome
Explain how reverse transcriptase is used to produce cDNA libraries
mRNA is extracted from cells and oligo(dT) primer is added
reverse transcriptase synthesizes complementary DNA (cDNA) strand, resulting in ds RNA/DNA hybrid
RNA in the hybrid is partially digested with RNAase
DNA polymerase I synthesizes a 2nd DNA stand
DNA ligase joins the gaps
what is PCR
polymerase chain reaction- a method for amplifying or copying target DNA sequences
what is needed for PCR
DNA target polymerase (taq) ligase exo/endonucleases primer-ssDNAs one for each strand, must be complementary to the opposite ends of the target sequence nucleotides- dNTPs mg-cofactor usually in buffer
List the steps of PCR
a single run is of 25-30 cycles, each cycle has 3 steps: denaturation, annealing and extension
1st cycle: DNA denatured at 95C
temp lowered to 55-65- allowing primers to anneal to complementary DNA sequences on template strands. temp raised 72C to allow taq pol to extend primers, synthesizes complements to template
end of 3rd cycle:
2 copies of target sequence created, 6 are longer, longer products will get diluted out.
why can taq polymerase withstand the heat in PCR
it is from archea bacteria that live in hot springs, it is very heat resistant
what is electrophoresis
a separation method that separates biological molecules by their sizes by migrating them through a stationary medium under the influence of an electric field
what is the role of the medium agarose in electrophoresis
it is a polymer that provides resistance to mol movement
List the steps of electrophoresis
DNA sample is loaded into the well
electrical current is applied
DNA migrates towards + anode
visualize with gel red to see how far DNA travelled
List the steps of nucleic acid hybridization
- NA in a sample separated by electrophoresis
- NA is denatured to prepare for hybridization as gel is running
- NAs transferred from gel to NA binding membrane
- membrane probed with an ssDNA dinucleotide
- the bound probe is detached
What charge to nucleic acids have
negative charge
what information can we learn from NA hybridization
if the NA is present (band or no band)
how large is the NA (how far did the band travel)
about how much of the NA is present (how thick is the band)
what is the difference between DNA sequencing by chain termination and PCR
only one of the 2 strands is sequenced in chain termination, only one primer is needed
what is the effect a dideoxynucleotide has on transcription?
it stops it because it has an H where the OH should be, DNA polymerase cant go past it
explain the steps of gene sequencing by chain termination
1- dNTPs and a small amount of ddNTP, primer, DNA polymerase and the DNA template strand are all placed together. each ddNTP has a different fluorescent color
2- synthesis starts mostly with normal dNTP but when ddNTP is inserted, the replication terminates
3-fragments are separated by capillary gel electrophoresis
4-fluorescence if the ddNTP terminating each strand is detected and a sequence is generated
Explain how third generation sequencing works
DNA pol located in a nanopore anchored to a solid substrate binds a ssDNA mol to be sequenced
DNA pol adds a tag to synthesize DNA
tag is cleared off each base and is added to the DNA strand
What is a knockout organism
an organism where a single gene has been “deleted” only possible with non essential genes
how are knockout organisms created
a targeting vector containing a mutated version of the gene of interest is transformed into embryonic stem cells growing in a culture.
homologous recombination in the embryonic cell replaces the gene of interest with the mutant
recombinant ES cells are then selected and microinjected into an embryo in the blastocyst stage.
embryos are implanted into surrogate
surrogate gives birth to chimeric offspring (some KO stem cells some normal)
chimeric animal is bred to generate homozygous KO animal
what does CRISPR-Cas stand for
clustered regularly interspersed short panel repeat sequences- cas is a protein
what is a CRISPR locus
consists of clustered repeats with non repetitive spacer sequences
where do spacer sequences come from in CRISPR
derived from portions of an invading genome from a prior invasion
Explain how CRISPR can edit a genome sequence
cas 9 is introduced into target cell along with ss guide RNA complementary to the genomic sequence.
cas 9 generates double stranded DNA break, the break is repaired and an insertion or deletion is generated.
what is genetic engineering
alteration of a cell/ organism genome
what is a genetically modified organism
carries a gene transferred from another species and is expressed to produce a protein product.
what is biotechnology
a use of biological organisms (or their cells/ molecules) to create a product or process that is useful to humans
explain how biotechnology can be used in agriculture
transgenic crops are created to resist herbicides and pests or can be nutritionally enhanced
explain how genetically modified animals are used in biotechnology
to study gene function, serve as animal models in human disease. can also be genetically altered to resist disease when the animal is used in food production
how are pharmaceutical products a part of biotechnology
therapeutic proteins are used to treat diseases like insulin
What is RFLP
restriction fragment length polymorphism are variations in the length of DNA fragments produced by restriction endonucleases
what is population genetics
it analyzes the amount and distribution of genetic variation amoung individuals in populations
what is a population
a group of individuals of the same species that live in a defined area and can breed
what factors can cause variation in population genetics
single nucleotide polymorphisms
microsatellites
what are SNPs
single nucleotide polymorphisms, differences in nucleotide pairs present at a single site in the genomes of 2 or more naturally occurring individuals
how common are SNPs in humans
1 in 500 to 1000 bps
what are microsatellites
repeats of a short sequence motif (2-6 bps long) sequence is repeated a certain number of times
how common are microsatellites in the human genome
very, there are more than 1 million
where is it possible to find microsatellites
anywhere in the genome, could be introns, exons, caps or tails
how can satellites be analyzed
through PCR to determine the number of repeas present, the higher up the bigger the molecule the more repeats
how is variation measured
genotype frequency allele frequency using the hardy wienburg law
what are the conditions for the hardy weinberg law to apply, what is the term for this
large random mating populations in the absence of evolutionary forces, hardy wienberg equilibrium
what factors influence changes in allele frequencies
non-random mating (inbreeding) natural selection mutation migration genetic drift genetic bottleneck effect
what is genetic drift
a small number of individuals originate from a new population of species
what is the genetic bottleneck effect
a large number of population undergoes drastic temporary reduction in numbers
what is gene therapy
the use of genes as therapeutic agents to cure disease or alleviate symptoms
what was the 1st genetic disease treated with gene therapy
SCID severe combined immunodeficiency disease, no functional T cells generated in body
setback was cancer in many patients
