CHAPTER 4-CRISPR Flashcards
Restriction enzymes
restriction enzymes, also known as restriction endonucleases cut DNA into smaller fragments in order to isolate the gene of interest.
RNA polymerase
an enzyme that copies the DNA strand to construct a pre-mRNA strand during transcription.
Ligase
: are enzymes that join DNA fragments by forming phosphodiester bonds, after the restriction enzymes cut DNA.
Cutting of DNA:
Restriction enzymes cut DNA into smaller fragments to isolate the gene of interest by cutting a specific recognition site and they cut by cleaving the phosphodiester bond of the sugar-phosphate backbone.
resriction enzymes are found in bacteira
What is a recognition site, is the recognition site of an an enzyme specific\, and 2 types of restriction enzymes and wha thtey do and compare the DNA in prokaryotes and eukaryotes
Recognition site is wheere the restriction enzyme cuts the DNA
The two types of restrtcion nezymes are:
sticky end restricton nezyme: when it cuts DNA, it creates a staggered cut, leaving overhanging, unpaired nucleotides.
Blunt end restriction enzyem: Cut in the middle of the recognition site and straight cut is madeleaving no overhanging nucloetides
Linear DNA is seen in the nucleus of eukaryotic organisms. Circular DNA is seen in the cytoplasm of prokaryotic cells, organelles like chloroplast and mitochondria.
Linear DNA always makes one extra cut eg, 1 cut resutls in 2 fragmetns but circualr 1 cut results in 1 fragment
Ppolymerase chain reaction(PCR), what is the purpose of PCR, and what is PCR used for. why can PCR be used for foresinic testing and paternity testing?
process involves making large amounts of DNA in a short amount of time.
The purpose of PCR is that it is used when there is not enough samples of DNA.
PCR once the DNA is amplified can be used for foresninc testing, paternirt testing and testing for genetic diseases.
PCR can be used for forensic testing because PCR can amplify DNA from tiny samples found at the crime scene eg. blood or hair for DNA fingerprinting or identifying individuals through genetic profiles.
PCR can be used for paternity testing to compare DNA from parents and children to confirm that the children are biological children of the father.
Stages of PCR, what happens to taq polymerase above 95 degrees,at 72 degrees and below 72 degrees
- Denaturation:
Sample DNA is heated to approx 95 degrees to break the hydrogen bonds of the strand, forming single stranded DNA. - Annealing:
The single stranded DNA is cooled down to approx 55 degrees. This allows the primers to anneal. These primers act as the starting region for the next stage. - Elongating stage:
The DNA is heated to 72 degrees which allows the Taq polymerase to work optimally. Taq polymerase binds to the primers and moves through the DNA strand. Free nucleotides are added to form a double stranded DNA molecule( - Repeat:
Stages 1-3 are repeated multiple times to create more copies of DNA.
note that Taq polymerase most optimal temperature is 72 degrees, at a temperature above 95 it will denature and not function, anything lower than 72 its function will decrease)
What is the role of polymerase( DNA and RNA polymerase0 and what is a primer and Taq polymerase and lastly, What is used to copy the DNA sample?
Polymerase are enzymes that add nucleotides to DNA or RNA, leading to genes being copied. A primer is needed to start this process
RNA polymerase copies the DNA strand to produce a pre-mRNA strand in transcription
DNA polymerase adds DNA nucleotides to form DNA in cell replicaiton.
A primer short segment of RNA or DNA that acts as the starting point for DNA synthesis.
-Taq polymerase is an enzyme used to amplify a DNA sequence by producing new strands of DNA by adding nucleotides to single stranded DNA template during the PCR process.
Primers, free nucleotides, and Taq polymerase.
GEL ELECTROPHORESIS what is it, what can it be used for and how can gel electrophreosisi be used for genetic testint and how can gel electrphoreisis be used to determine a genetic disase
a technique used to measure the size of DNA fragments by placing the fragments of DNA cut by restriction enzymes in a gel and using electrical current to separate DNA based on size.and is used after restriction enzymes and PCR,
Gel electrophoresis can be used for:
Forensic analysis
DNA profiling
Paternity testing
Test for genetic disorders
gel electrophoresiscan e used is to confirm a genetic disorder and determine if a person has a gene which predisposes them towards developing a disease such as breast cancer. Once a DNA sample is obtained, gel electrophoresis can be used to determine if the disease causing allele is present.
The gel in gel electrphoreiss acs as a sieve for the movement of DNA
what is applied to the gel in gel electrophoreiss, since DNA is negatively charged whihc electorde will it movet owards and why, what travels fast smaller or larger fragments of DNwhat is a standard latter
an electriv current is applied to the gel
The negative end is where he DNA is loaded
The positive end is where the DNA moves towards
DNA is negatively charged and will move towards the positive terminal because opposite charges attract nadsince DNA is negatively charred it is pulled to the positive terminal.
Smaller fragments of DNA will travel quicker than larger fragments of DNA they experience less resistance from the gel matrix, allowing them to travel more easily through the pores compared to larger fragments.
The gel acts as a sieve to allow for the movement of DNA.
A standard ladder is known DNA sizes to compare the sample
Steps of Gel electrophoresis and how to read gels.
The negative end is where the DNA is loaded
The positive end is where the DNA moves towards
- DNA samples are placed in the wells of the gel using a pipette. Lane 1 of the gel usually includes a standard ladder of known sizes. The samples are always places of the negative end.
2.After loading the wells, a buffer solution is added to the gel. This helps conduct the electrical current. Positive and negative electrode is connected and electricity is turned on.
3.After 30 minutes, DNA fragments move from the negative end of the gel to the positive end. This is due to the DNA being negatively charged due to the phosphate group.
Smaller DNA fragments move faster and further from the wells.
4.DNA is now separated based on fragment size. A dye/UV stain is usually added to see the bands of the gel.
What is cystic fibrosis, and how can it be detected using gel electorphoreis
Cystic fibrosis(CF) involves the deletion of 3 nucleotides in the CFTR gene. Cystic fibrosis is a recessive trait which means individuals must have 2 allele to be impacted by CF.
Sample DNA is collected after birth,amplify the region of the CFTR resticton enzymes isolate gene of itnerest gene using PCR to create DNA fragmenets and seperate the gels via size via electrophoreiesis then compare the band pattern to a normal sample, a differnet pattern indiicates a mutatoon in the CFTR gene suggestingcystici fibrosis
What is DNA profiling, what is the purpose of DNA profilign and what are short tandem repeats and what are the implications of DNA profiling.
DNA PROFILING:
DNA profiling is the process of identification using genetic information, DNA profiling is also known as DNA fingerprinting.
Purpose of DNA profiling:
To identify criminals in forensic investigation
Determine is families are biologically related for things like custody
Identifying bodies
Short tandem repeats(STRs) are repeating nucleotides within the coding region of a gene.
Social implications of DNA profiling:
benefit- helpful to indentify and convict perpreators ofa crime
consequence- privary isses- who will have access to the genetic data
Ethical implications of DNA profiling:
benefits:
Can determine lineage and assist with organ donors and patients
Personal Data may be leaked, results are not always reliable and DNA may be contaminated.
What is bacterial transfomration,
The techniques for bacteria to take up a plasmid are/bacterial transformatioj,
Q: What are plasmids?
Why is advantagoeus to use plasmids as vectors
is the process where a plasmid(circular dna) will enter a bacteria, resulting in it acquiring new geneitc traits, this bacteria is said to be transofmred
- Heat shock: Heat shock is where bacterial cells and recombinant cells are placed in an ice cold solution containing calcium ions. The calcium ions help make up the plasma membrane more permeable. The solution is then heated to 37-47 degrees then cooled down in ice to allow the plasmid to enter the bacterial cytoplasm.
2.Electroporation: an electrical current is passed through the solution allowing the plasmid to enter via the bacterias cytoplasm.
A: Plasmids are circular DNA molecules that can replicate independently of the bacterial chromosome.
Why is advantageous to use plasmids as vectors
Plasmids are small, hence they are easy to manipulate
Plasmids carry a wide range of restriction enzymes
Recombinant plasmids replicate independently once they are inserted into their host cell.
What are the steps in making a recombinant plasmid
1.Restriction endonucleases are used to cut, e.g. the plasmid
- DNA ligase joins the DNA sequences into the plasmid.
- The plasmid is inserted by heat shock into the bacteria which is transformed OR each plasmid is inserted into separate bacteria.
- Antibiotic selection or another suitable method (e.g. insulin genes inserted next to a gene coding for beta-galactosidase protein) is used to determine success.
- Seletcion of bacteria
