Quiz 8 Flashcards
Polymerase chain reaction
DNA replication in vitro that amplifies a target DNA sequence present in very small quantities (PCR product)
Ingredients needed for PCR
DNA with target sequence, two oligonuclrotide primers (synthesized in a lab), the four nucleotides, DNA polymerase
Three steps of PCR reaction
Separating the template strands by heating to almost boiling so they denature, annealing the primers to the template DNA strands by lowering the temperature tk about 60 celsius, and synthesizing the DNA by raising to 72 celsuis
Extension
Extending the primers; synthesizing the complementary strands
The first cycle of PCR reaction makes:
Two copies of target DNA sequence
A PCR experiment consists of:
20-30 cycles (30 cycles generated over a billion copies of one molecule of a target sequence)
Amount of target DNA needed:
One cell!
Heat stable DNA polymerase
Specifically taq, from a bacteria that lives in high temperatures
Limitations of PCR
We need to know sequence of target, contamination, long DNA sequences can’t be amplified
Applications of PCR
Cloning, identification of DNA in samples, genetic testing, and examining gene expression in cells
Gene targeting
Altering the DNA of a living organism by introducing a gene using homologous directed repair
Gene editing
The use of specific enzymes to edit the sequence of a gene in an organism
Knockout
An organism that has a targeted disruption of a gene (i.e. it has been made to not work)
Why knockouts are produced:
So we can learn about the function of a gene; if both alleles are not functioning then the phenotype of the organism will be different compared to an organism that has one or two wild-type alleles for that gene
Process of producing a knockout mouse:
Constructing a target vector that contains the gene of interest mutated using a neomycin (disrupts and acts as selectable marker) and the flanking noncoding regions of the gene; target vector then undergoes homologous recombination (i.e. replaces target gene)
Introducing target vector to embryonic stem cell harvested from the inner cell mass of a blastocyst; if ES cell takes up vector, then homologous recombination can occur (usually only replaces wild-type gene on one chromosome)
Injecting recombinant ES cell into mouse embryo and implanting it into a surrogate mother, which will birth chimeras (some will contain target disruption, others won’t) which can then be bred with normal mice to produce heterozygous offspring for the targeted disruption, then bread the hetrozygous mice to produce a homozygous offspring
Neomycin
Inserted into gene of interest to be used for a knockout; disrupts gene and serves as selectable marker using antibiotics
Homologous recombination
Replacement of target gene in a chromosome
Conditional knockout
Knockout whose targeted disruption can be controlled in time, such as if the target gene is necessary for embryonic development; gene is disrupted by researchers at a particular time in a particular organ or tissue
Cre-lox system for a conditional knockout
Construct a target vector that contains gene of interest flanked by loxP sequences, follow the same approach as with a normal knockout for breeding, breed these mice with those that have been modified to have the Cre gene in their germ cells (Cre is controlled by a promoter that can be activated by a specific hormone or only in a specific tissue)
Offspring produced that contains the gene of interest flanked by the loxP sequences and the Cre gene with its promoter; the Cre gene is activated to create Cre recomibnase, which recognizes the loxP sequences and recombines them to delete the gene between them
Transgenic animals
An organism that has an added gene (transgene) in its genome; opposite of a knockout
Why transgenic animals are produced:
To learn the function of a gene or espress a gene from one organism in another organism
Process of producing a transgenic organism
Similar process to knockout but no need for target vector cause it doesn’t have to be in a particular place in the chromosome
Examples of transgenic animals:
Green flourescent mice, big mice with the growth hormones of rats
Gene editing
Use of specific enzyme to edit the sequence of a gene in an organism, thus modifying its sequence by removing, correcting, or replacing a defective gene or parts of a gene (using enzymes that cut DNA in a specific manner)
General features of CRISPRA-Cas
Cas9 protein cuts DNA at specific sequences that have a PAM sequence next to them; a guide RNA directs the Cas9 to the exact location of the cut, and a replacement sequence is provided and inserted where the cut took place; initially found in bacteria as a defense against viruses/plasmids
Defense mechanisms developed by bacteria:
Innate immunity (nonspecific mechanisms that are not targeted) and adaptive immunity (specific mechanisms, such as CRISPR-Cas, that are targeted against particular phages after the bacteria is exposed to and can recognize the phage)
Components of CRISPR-Cas mechanism
CRISPR, a locus in the bacterial genome composed of a leader sequence, short palindromic repeats, and spacer sequences (short sequences derived from phages, flanked by repeats)
CRISPR-associated (Cas) genes that are located next to CRISPR locus and encode different proteins including the enzymes that cuts phage DNA
Three steps of CRISPR-Cas mechanism
Spacer acquisition (a phage infects and injects its DNA, which is then cut into small fragments called protospacers which are then inserted into CRISPR locus and become new spacers in it (newer spacers located closer to leader sequence)
crRNA biogeneis occurs; the CRISPR locus is transcribed to produce pre-crRNA, which is processed into small mature crRNAs (spacers flanked by repeats)
During a new infection by the same oahgez the crRNA guides a CAS nuclease to injected phage DNA, which then cuts cuts it
Protospacers
Cut phage DNA pieces
Spacer DNA serves as:
Molecular memory of the previous phage infection
Experimental evidence supporting the “molecular memory” theory:
If spacer sequences are experimentally deleted, the bacterium loses its ability to remember the phage and is sensitive to it, and vice versa; furthermore, phages respond by mutating so the spacers in CRISPR locus don’t match the phage DNA anymore
CRISPR-Cas in streptococcus pyogenes
Spacers acquired when Cas9 cuts injected phage DNA next to PAM sequence and Cas1/Cas2 complex inserts the spacers into the CRISPR locus, then a tracrRNA molecule binds to the repeats of the pre-crRNA to form dsRNA, then to Cas9 and an RNAse cuts the pre-crRNA to make mature crRNA and tracrRNA complexes
The repeat region of crRNA is bound by tracrRNA to make dsRNA; the spacer forms a single stranded portion of crRNA and recruits Cas9 to matching phage DNA that has a PAM sequence (HNH domain that cuts the ohage DNA and is complementary, and the RuvC domain that cuts the phage and is not)
PAM sequences provide a way for CRISPR-Cas system to distinguish self from non-self, as only the phage that contains the PAM sequence is cut
Single guide RNA
Hybrid crRNA/tracrRNA molecule that can be designed to match any twenty nucleotides of a target sequence, and only requires the sgRNA and Cas9 to cut
CRISPR-Cas9 genome editing
Using homology-directed repair mechanism, which is a pathway that repairs double stranded breaks using sister chromatin; we can trick HDR by using a donor template that contains a piece of DNA we want to insert into the genome
Add Cas9, an sgRNA that matches the target DNA, a donor template with the desired DNA
CRISPR-Cas infidelity during gene editing
Cas9 occasionally cuts DNA not intended to be cut because sgRNA also matches other pieces of DNA, and it’s part of a bacterial system that combats phages constantly mutating so it’s a little sloppy; solutions include designing sgRNAs carefully and mutating Cas9 so it’s more accurate
Two conditions for treating a genetic disease with gene therapy
The genes causing the condition must have been identified and cloned; accessible (blood cells), and correct cells must be able to be targeted
Ex vivo vs in vivo gene therapy
Ex vivo involves cells derived from patient that are treated in a lab and returned, whereas in vivo invites Introducing therapeutic DNA directly into affected cells in body
Viral vectors used to deliver therapeutic genes
Genetically engineered to carry the gene of interest, infect cells, and deliver gene to cells, while bot causing disease
Adenovirus vectors
Very commonly used, DNA viruses that can carry relatively large therapeutic genes but have antibodies produced against them causing immune reactions
Lentivirus vectors
RNA retorvirus that have their replication/disease-causing genes removed and genes of interest added to infect the human cells and integrate (ex: HIV)
Advantageous long term expression, large pieces of genetic material, can infect non-dividing cells, but integration is random and as such can lead to insertional mutations
Severe combined immunodeficiency disease
First disease treated with gene therapy
Nonviral delivery of therapeutic genes
Both in and ex vivo, such as chemically assisted transfers, lipsomes, and gene pills; just have to ensure that DNA can cross the plasma membrane
Adeno-associated virus (AAV) vectors
Often used in gene therapy trials, DNA viruses that are nonpathogenic and infect cells to make episomes; but do not replicate when cells divide
Using stem cells to deliver therapeutic genes
Viral or nonviral vectors deliver therapeutic genes via stem cells in vitro either with genetic modifications or differentiated in vitro and matured before given to patient
Issues with vectors used in gene therapy
Adverse immune responsez insertional mutations, many human genes are too big for vectors, integration of retroviral DNA only occurs when cells are dividing, and risk of an infectious virus being created
Immunotherapy
Using patient’s own immune cells to kill tumors
Hematopoietic stem cells
Stem cells found in bone marrow; easily accessible, taken from patient so little risk for immune rejection, replicate quickly in vitro, fairly long lived, and differentiate into both red and white blood cells
Gene editing approaches to gene therapy
Two gene editing methods, ZFNs and TALENs, have been used to replace genes such as to cure the blistering disease
RNA based approaches to gene therapy
Small pieces of RNA can be used to silence gene expression; antisense oligonucleotides can stop mRNA from being translated, while RNAi uses signals and other proteins to degrade mRNA or stop it from being translated
