DNA Manipulation Flashcards
What are endonucleases?
Enzymes that break the phospodiester bonds between two nucleotides in a polynucleotide chain
What are estriction endonucleases?
enzymes that acts like molecular
scissors to cut nucleic acid strands
at specific recognition sites. Also
known as a restriction enzyme
What is a sticky end?
the result of a staggered cut through doublestranded DNA by an endonuclease
resulting in overhanging nucleotides
What is a blunt end?
the result of a straight cut across the double-stranded DNA by an endonuclease resulting in no overhanging nucleotides
What are ligase?
enzymes that join two fragments on DNA or RNA together
What are polymerases?
they add nucleotides to DNA or RNA, which can lead to copying entire genes
What is a primer?
a short, single strand of nucleic acids that acts as a starting point for polymerase
enzymes to attach
What is a bacteriophage?
a virus that infects prokaryotic organisms
What is CRISPR-Cas 9
a complex formed
between gRNA and Cas9 which
can cut a target sequence of
DNA. Bacteria use this complex
for protection from viruses and
scientists have modified it to
edit genomes
What is a spacer?
short sequences of DNA obtained from invading bacteriophages that are added into
the CRISPR sequence
What is a protospacer?
a short sequence of DNA extracted from a
bacteriophage by Cas1 and Cas2, which has yet to be incorporated into the CRISPR gene
What does PAM stand for? And what is it?
PAM = protospacer adjecent motif. It’s a sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9. It uses NGG
What is guide RNA (gRNA)?
RNA which has a specific sequence determined by CRISPR to guide Cas9 to a specific site
What is single guide RNA? (sgRNA)
sgRNA is just the name we use when CRISPR-Cas9 is used by scientists as a gene-editing tool
What is gene knockout?
a technique in gene editing where scientists
prevent the expression of a target gene to understand its function in an organism
What is gene knock-in?
a technique in gene editing where scientists substitute or add nucleotides in a gene
What is polymerase chain reaction?
a laboratory technique used to produce many identical copies of DNA from a small initial sample
What is Taq Polymerase?
a heat-resistant DNA polymerase enzyme isolated from the bacteria Thermus aquaticus, which amplifies a single-stranded DNA molecule by attaching complementary
nucleotides
What is the first stage of Thermal cycler?
Denaturation – DNA is heated to approximately 90–95 °C to break the hydrogen bonds between the bases and separate the strands, forming single-stranded DNA
What is the annealing process in a thermal cycler?
the single-stranded DNA is cooled to approximately 50–55 °C to allow
the primers to bind to complementary sequences on the single-stranded DNA.
What is the elongation process in a thermal cycler?
the DNA is heated again to 72 °C, which allows Taq polymerase to work
optimally. Taq polymerase binds to the primer, which acts as a starting point, and begins synthesising a new complementary strand of DNA.
What is gel electrophoresis?
a technique that separates DNA fragments
based on their molecular size
What are short tandem repeats (STR) ?
short, repeated sequences of nucleotides found in the noncoding regions of nuclear DNA
