Enzymes that manipulate DNA Flashcards
Define Endonuclease
An endonuclease is an enzyme that breaks the phosphodiester bond between two nucelotides in a polynucleotide chain.
Define a recognition site
A recognition site is a specific target sequence of DNA upon which restriction endonucleases do their job.
Define Restriction enzymes/endonucleases
Restriction endonucleases are any enzymes that acts like ‘molecular scissors’ to cut nucleic acid strands at specific recognition sites.
Distinguish the difference between sticky and blunt end cuts of DNA.
Sticky end cuts refers to the result of a staggered cut through double-stranded DNA by an endonuclease resulting in overhanging nucleotides. Blunt end cuts refers to the result of a straight cut across the double-stranded DNA by the endonuclease resulting in no overhanging nucleotides.
Define Ligases
Ligases refer to an enzyme that joins molecules, including DNA or RNA, together by catalysing the formation of phosphodiester bonds.
Define a primer
A primer is a short, single strand of nucleic acids that acts as a starting point for polymerase enzymes to attach.
Define a bateriophage
A bacteriophage is a virus that infects prokaryotic organisms.
Explain what CRISPR-Cas9 is
CRISPR-Cas9 is a complex formed between gRNA and Cas9 which cuts a target sequence of DNA. BActeria use this for protection and used for editing genomes.
Define spacers
Spacers are short sequences of DNA obtained from invading bacteriophages that are added into CRISPR sequence.
Define protospacer adjacent motif (PAM)
PAM is a sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9.
Define guide RNA (gRNA)
Guide RNA is RNA which has a specific sequence determined by CRISPR to guide Cas9 to a specific location or site.
Define single guide RNA (sgRNA)
Single guide RNA is guide RNA utilised by scientists to instruct Cas9 to cut a specific site when using CRISPR in gene editing.
Identify the steps of using CRISPR-Cas9 in gene editing
- sgRNA is created with a complimentary spacer to target DNA wanted to be cut. 2. Cas9 enzyme is obtained with the appropriate target PAM sequence. 3. Cas9 and sgRNA are added together and mixed to bind together and create CRISPR-Cas9 complex. 4. sgRNA-Cas9 mixture is then injected into a specific cell. 5. Cas9 finds target PAM sequence and checks to see whether the sgRNA aligns. 6. Cas9 cuts the selected sequence of DNA. 7. DNA has a blund end cut that cell will try to repair.
Identify the applications of CRISP-Cas9 technology.
Some applications of this technology include: Research, Dealing with diseases and Agriculture.
Define Differentiation
Differentiation is the process of which cells can develop specialised characteristics, typically transforming from one cell type to another specialised cell type.
Define the Polymerase Chain Reaction (PCR)
The polymerase chain reaction is a technique used to produce many identical copies of DNA from initially a smaller sample.
Define Taq Polymerase
taq polymerase is a heat-resistant DNA polymerase enzyme which amplifies a single-stranded DNA molecule by attaching complementary nucleotides.
Identify the steps of Taq Polymerase
The steps of taq polymerase are Denaturation, Annealing and Elongation.
Explain the process of Denaturation
DNA is heated up to approximately 90-95 degrees to break the hydrogen bonds between the bases and separate the strands, forming single-stranded DNA.
Explain the process of Annealing
The single-stranded DNA is cooled to approximately 50-55 degrees to allow for primers to bind to complimentary sequences on single-stranded DNA.
Explain the process of Elongation
The DNA is heated again to 72 degrees, which allows for Taq polymerase to work optimally. Taq polymerase binds to primer, acts as a starting point, and begins synthesising a new complimentary strand of DNA.
Define Gel electrophoresis
Gel Electrophoresis is a technique that separates DNA fragments based on their size.
Define a well
A well refers to an indent in the gel into which a DNA sample is loaded into.
Define a standard ladder
A standard ladder refers to a mixture of DNA fragments of known length that are used to infer the size of the fragments to move through.
Define agarose gel
Agarose gel refers to a sponge-like gel that contains pores for DNA fragments to move through.
Define buffer
It refers to a solution that carries electrical currents through the agarose gel.
Define a base pair (bp)
A base pair is a unit of measurement that corresponds to one nucleotide.
Define DNA profiling
DNA profiling is the process of identification on the basis of an individuals genetic information.
Define heterozygous
Heterozygous refers to having different alleles for the same gene on homologous chromosomes.
Define homozygous
Homozygous refers to havign identical alleles for the same gene on homologous chromosomes.
Define Short Tandem Repeats (STR)
short tandem repeats are short, repeated sequences of nucelotides found in the non-coding regions of nuclear DNA.
Define a plasmid
A plasmid is a small, circular loop of DNA separate from the chromosome, found in bacteria.
Define Recombinant plasmids
recombinant plasmids refer to a circular DNA vector that is ligated to incorporate a gene of interest
Define the process of bacterial transformation
Bactral transformation is the process by which bacteria take up foreign DNA from their environment. It is used to introduce recombinant plasmids into bacteria.
Define plasmid vector
Plasmid vectors refer to a piece of circular DNA that is modified to be an ideal vector for bactral transformation experiment.
Define antibiotic resistance gene
The antibiotic resistance gene refers to the gene which has resistance to antibiotics.
Define reporter gene
The reporter gene refers to the gene with ann easily identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest.
Define genetic engineering
Genetic engineering refers to the process of using biotechnology to alter the genome of an organism. It involves genes being silenced, inserted into a genome, removed from a genome or altered by replacing nucleotides.
Define a GMO
A genetically modified organism refers to an organism with genetic material that has been altered using genetic engineering tech.
Define cisgenic organisms
A cisgenic organism refers to a GMO thatc contains foreign genetic material from a sexually compatible donor organism, typically from same species.
Define transgenic organisms
Transgenic organisms refer to a GMO that contains foreign genetic material from a separate species (or recombinant DNA from the same species that has been manipulated before introduction).
Explain the use of GMO in agriculture
through the use of agriculture, the applications of GMO’s include: increased crop productivity and increased disease resistance of the crop.
Define Transgene
Transgene refers to a gene that has been artificially introduced into the genome of a separate organism.
Identify the issues surrounding the use of GMO’s
Some issues surrounding the use of GMO’s include: Biological implications - GM crops may lose effectiveness, widespread use of GM crops may result in loss of genetic diversity, Social implications - costly for farmers, strict packaging and marketing guidelines, Ethical implications - Some people consider GMO’s to be unnatural, people believe genetically modifying animals is inhumane and wrong.
