Recombinant DNA Technology Flashcards
Briefly explain the topic of Myostatin transgene animals
Create more muscle causing animals to appear more muscular and buff
Define Molecular cloning
A molecule, cell, or organism that was produced from another entity
Provide an example of Reproductive Cloning
Example includes Dolly the Sheep
Gene cloning was made possible by the discovery of what?
- Restriction Enzymes
- Plasmid DNA Vectors
Define Restriction Enzymes
DNA cutting enzymes (molecular scissors)
Define Plasmid DNA Vectors
- Circular form of self-replicating DNA
- Can be manipulated to carry and clone other pieces of DNA
Explain Restriction Enzymes (deeper definition)
- Primarily found in bacteria (they use these for defense)
- Cut DNA by cleaving the phosphodiester bond that joins adjacent nucleotides in a DNA strand
- Bind to, recognize, and cut DNA within specific sequences of bases called a restriction site
- Each restriction site is a PALINDROME– reads same forward and backward on opposite strands of DNA
- There are 4 or 6 bp cutters because they recognize restriction sites with a sequence of 4 or 6 nucleotides
What are some examples of restriction enzymes with the way they cut DNA (2 examples)
- Some cut DNA to create DNA fragments with overhanging single-stranded ends called “sticky” or “cohesive” ends
- Some cut DNA to generate fragments with double-stranded ends called “blunt” ends
Which type of cutting is preferred with Restriction enzymes?
- The one that produces the STICKY ENDS
- Preferred for cloning because DNA fragments with sticky ends can be easily joined together because they base pair with each other by forming weak hydrogen bonds
Explain what Plasmid DNA is
- Small extrachromosomal DNA that are circular pieces of DNA found primarily in bacteria.
- Extrachromosomal definition since they are in the cytoplasm
- Small approx. 1 to 4 kb
- Can replicate INDEPENDENTLY of chromosome
- Can be used as vectors - pieces of DNA that can accept, carry, and replicate other pieces of DNA
In 1975, NIH formed the RAC (Recombinant DNA Advisory Committee) based on results from Asilomar meeting.
What is the purpose? What was published?
- Purpose of RAC: evaluate recombinant technology and guidelines for research
- 1976 RAC published set of guidelines for working with recombinant organisms
Explain Blue-white selection
- DNA is cloned into the restriction site in the lacZ gene
- When it is interrupted by an inserted gene, the lacZ gene cannot produce functional Beta gal
- When Xgal (artificial lactose) is added to the plate, if functional lacZ is present = blue colony
- Non-functional lacZ = white colony = clone = genetically identical bacterial cells each containing copies of recombinant plasmid
What is Selection in terms for recombinant DNA technology
Process designed to facilitate the
identification of recombinant bacteria while preventing the growth of non-transformed bacteria and bacteria that contain plasmid without foreign DNA
Explain Electroporation
Apply brief pulse of high voltage electricity to create tiny holes in the bacteria cell wall that allow the DNA to enter
Transformation of Bacterial cells is very INNEFICIENT. However, explain the process for inserting foreign DNA into bacteria.
- Treat bacterial cells with calcium chloride
- Add plasmid DNA to cells chilled on ice
- Heat the cell and DNA mixture
- Plasmid DNA enters bacterial cells and is replicated and express their genes
Explain Antibiotic selection
plate transformed cells on plates containing different antibiotics to identify recombinant bacteria and non-transformed bacteria
What 2 hormones were the 1st human protein expressed via recombinant techniques
Insulin and then Growth Hormone
What was source of growth hormone prior to recombinant technology?
Pituitary glands of Cadavers or just from other animal/humans
What are the 5 Practical Features of DNA Cloning Vectors?
- Size – small enough to be separated from chromosomal DNA of host plasmid
- Origin of replication (ori) – site for DNA replication that allow plasmids to replicate independently from host
chromosome
* Copy number: number of plasmids in the cell (normally small but plasmids have high copy numbers) - Multiple cloning site (MCS) – recognition sites for several restriction enzymes in which insert is cloned into
- Selectable marker genes – allow to select for transformed colonies
- DNA sequencing primers
Why is Yeast artificial chromosomes (YAC) a good vector?
- Is miniature version of eukaryotic
chromosome containing ori or rep; two telomeres; selectable markers; centromere that allows replication of YAC and segregation of daughter cells - Best for cloning very large DNA inserts from 200 kb to 2 megabases
- Were used for human genome project
– Small plasmids grown in E coli and introduced to yeast cells (S. cervisiae)
Why is Ti vector a good vector?
- Naturally occurring plasmids isolated from the bacterium that is a soil plant pathogen causing disease in plants
– When the bacteria infects plant cells, the T DNA from the Ti plasmid inserts into the host chromosome
– T DNA codes for auxin hormone that weakens plant cell wall and infected plants divided and enlarge to form a tumor (gall)
– Scientists use Ti vectors to deliver genes to plants by removing toxic gene for auxin
Was Self-replicating life with s100% synthetic DNA created?
YES!!!
What is the minimum amount of genes to sustain cell replicating lifeform
microplasma has around 400 genes to sustain cell-replicating life
What are DNA Libraries
- Collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host
– Screened to pick out different genes of interest
What are Two types of DNA Libraries
- Genomic DNA libraries
- Complementary DNA libraries (cDNA libraries)
Note: cDNA is reverse transcribed from mRNA
What are Genomic Libraries
- Chromosomal DNA from the tissue of interest is isolated and digested with a restriction enzyme which produces many fragments that include the entire genome
– Vector is digested with same enzyme
– DNA ligase is used to ligate genomic DNA fragments and vector DNA
– Recombinant vectors are used to transform bacteria and theoretically each bacteria will contain a recombinant plasmid
What are disadvantages of genomic libraries
- Introns are cloned in addition to exons;
– Majority of genomic DNA is introns in eukaryotes so majority of the library will contain non-coding pieces of DNA - Many organisms have very large genome, so searching for gene of interest is difficult
- Time consuming!
cDNA libraries need to make double stranded DNA from mRNA. HOW?
a. enzyme reverse transcriptase catalyzes synthesis of complementary single stranded DNA from mRNA
i. Called complementary DNA (cDNA) because it is an exact
copy of the mRNA
b. mRNA is degraded either with an enzyme or alklaline solution
c. DNA Pol is used to synthesize second strand of DNA to create
double stranded cDNA
cDNA libraries cont. explanations
- mRNA from tissue of interest is isolated
- Need to make double stranded DNA from mRNA
- Short linker double stranded DNA sequences which contain
restriction enzyme recognition sites are added to the ends of the
cDNA - Cut with restriction enzyme, cut vector with same enzyme, ligate
fragments to create recombinant vectors - Then transform bacteria with recombinant vectors
What are the 3 advantages of cDNA libraries over genomic libraries
- Collection of actively expressed genes in the cells or tissues from
which the mRNA was isolated - Introns are NOT cloned
- Can be created and screened to isolate genes that are primarily
expressed only under certain conditions in a tissue
Assume that a gene involved in increased muscle mass is
expressed when the muscle cells are exposed to growth hormone.
What would be the source of the cDNA library: muscle cells or
muscle exposed to growth hormone?
What is a disadvantage of cDNA libraries over genomic libraries
Can be difficult to make the cDNA library if a source tissue with
an abundant amount of mRNA for the gene is not available
Provide brief process of PCR
- Target DNA to be amplified is added to a tube, mixed with
nucleotides (dATP, dCTP, dGTP, dTTP), buffer, and DNA
polymerase. - Paired set of Forward and Reverse Primers are added –
short single-stranded DNA oligonucleotides (20–30bp long)
– Primers are complementary to nucleotides flanking opposite
ends of target DNA - Reaction tube is placed in an instrument called a
thermocycler
What are the 2 advantages of PCR?
- Can amplify millions of copies of target DNA from small amount of starting material in short period of time
- To calculate the number of copies of target DNA starting with 1 molecule of DNA use this equation
2N in which N represents number of PCR cycles
Assume you want to do 25 PCR cycles to amplify your DNA insert, how many copies of DNA will you have at the end of your PCR?
2^25 copies
Specific # = 33,554,432 copies
In Agarose Gel Electrophoresis, is the migration distance inversely proportional to size of DNA fragment?
YES!!!
- Large fragments migrate slowly; smaller fragments migrate faster
– Tracking dye is added to the samples to monitor DNA
migration during electrophoresis
– DNA can be visualized after electrophoresis by the
addition of DNA staining dyes - Ethidium bromide: intercalate between DNA base pairs and it
fluoresces under ultraviolet light - Then a picture can be taken to document the gel results
How does EtBr work?
Intercalate between DNA base pairs and it fluoresces under ultraviolet light
Explain Restriction mapping gene structure
– Cut cloned gene with restriction enzymes to pinpoint location of the cutting sites
– Knowing restriction map is useful for making clones of small pieces of the DNA which is called subcloning
– These small pieces of DNA can then be sequenced
What is the protocol of restriction mapping?
a. Digest DNA with single or double restriction enzymes
b. Separate DNA fragments via agarose gel electrophoresis
c. Arrange fragments in order to make map of restriction sites
Explain Fluorescence in situ hybridization (FISH) and procedure (long answer)
Chromosome Location and Copy Number
– Identify which chromosome contains a gene of interest
Procedure:
* Chromosomes are isolated from cells and spread out on glass
microscope slide
* DNA or RNA probe for gene of interest is labeled with fluorescent nucleotides and incubated with slides
* Probe will hybridize with complementary sequences on
chromosomes on slide
* Slide is washed and exposed to fluorescent light
* Wherever probe has bound to the chromosome, it is illuminated to indicate the presence of the probe binding
* Do karyotype to determine which chromosome shows fluorescence
What is FISH used for?
- Analyze genetic disorders
- Determine which cells in a particular organ are expressing the particular mRNA
Understand Southern vs Northern vs Western Blot analysis. When are they used?
SNOW DROP
- Southern = DNA
- Northern = RNA
- Western = Protein
Understand Real time or quantitative (qPCR)
- Can quantify amplification reactions as they occur in real time
- Need special thermal cyclers that
se a laser to scan a beam of light through the top or bottom of each PCR reaction - Each reaction tube contains EITHER a dye containing probe or DNA binding dye that emits fluorescent light when illuminated by the laser
- Light emitted by the dyes correlates with amount of PCR product amplified
- Light is captured by the detector which relays info. to the computer to provide readout on amount of fluorescence
- Readout is plotted and analyzed to quantify the number of PCR products produced after each cycle
Explain Taqman probes
- Complimentary to specific regions of target DNA between forward and reverse primers for PCR
- Contain two dyes
- reporter located at 5’ end of probe and can release fluorescent light when excited by the laser and other dye is quencher which is attached to 3’ end of probe
Explain SYBR green
- Binds double-stranded DNA and as more double-stranded DNA is copied with each round of qPCR there are MORE DNA copies to bind SYBR Green which INCREASES the amount of fluorescent light emitted
T/F: Gene microarrays enable researchers to study ALL of the genes expressed in a tissue very fast
TRUE!!!
Microarray (gene chip) is created with use of small glass microscope slide where what briefly occurs?
Single stranded DNA molecules are spotted on the slide using an arrayer (computer controlled robotic arm) which fixes DNA (multiple copies of cDNA) at different spots on the slide which is recorded by a computer
When is it common to do microarray?
- When you compare two different conditions with 2 different colored dyes (one for treatment and other for control condition)
- Laser is scanned at different wavelengths for each probe and then the images are overlaid to make direct comparisons between the treatment and control
- Example study gene expression difference between cancer cells and normal cells to look for genes possibly involved in cancer progression
- Results of such studies can possibly lead to new drug therapies to combat cancer and other diseases
What is the procedure for a Gene microarray
– Extract mRNA from a tissue of interest and cDNA is synthesized from mRNA and labeled with fluorescent dye
– Labeled cDNA is incubated overnight with the array where it hybridizes with different spot on the array that contain complementary DNA sequences
– Can have over 10,000 spots of DNA
– Array is washed and scanned by a laser that causes cDNA hybridized to array to fluoresce
– Fluorescent spots reveal which genes were regulated and Intensity of fluorescence indicates relative amount of gene expression
Define TAL
Transcription Activator-Like
Define RVD
Repeat-variable di-residue
Double Strand Break Repair involved NHEJ and HR. Define them!
- NHEJ: Non-homologous End Joining
- HR: Homologous Recombination
Explain CRISPR
- Clustered Regularly Interspaced Palindromic Repeats (CRISPR) found in 40% of bacteria loci and 90% of archaea
- Prokaryotic immune system that
confers resistance to foreign genetic elements such as plasmids and phages, and provides a form of acquired immunity. - Segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a bacterial virus or plasmid
What are similar between CRISPR, Zinc Finger Nuclease, and TALEN
All are gene-editing technology that induce double-stranded breaks to target and modify specific DNA sequences
Define Cas 9
CRISPR associated protein 9 a nuclease which are specialized for cutting DNA
How many different types of CRISPR/CAS exists
type 1, type 2, and type 3
What is gRNA?
- Guide RNA
- A construct/chimera of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA)
Define PAM (not from the office)
- Protospacer adjacent motif with sequence
- Ex.) NGG means (any, guanine, guanine)
CRISPR/Cas as Immune System of Bacteria steps
- Acquisition of foreign DNA
- Synthesis and maturation of CRISPR RNA (crRNA) followed by formation of RNA-Cas nuclease protein complexes
- Target recognition by crRNA and destruction of foreign DNA by Cas nuclease cleavage
What are problems with CRISPR
- Off-target effects such as cleaving unintended DNA
- Delivery to cells in live humans tissues
- Variable DNA cleavage efficiencies
- Inter- and Intrachromosomal rearrangement
