BIOCHAP4 Flashcards
Endonucleases refer to
a broad range of enzymes responsible for cutting strands of DNA
Recognition site:
specific target sequence of DNA upon which restriction endonucleases act.
Restriction endonuclease:
any enzyme that acts like molecular scissors to cut nucleic acid strands at specific recognition sites.
endonucleases - When these enzymes target specific recognition sites, they are known as
restriction endonucleases.
endonucleases - To ‘cut’ the DNA..
enzymes cleave the phosphodiester bond of the sugar-phosphate backbone that holds DNA nucleotides together.
Restriction endonucleases are often sourced from
bacteria, produced as a defence mechanism against invading viral DNA.
Endonucleases either create sticky ends or blunt ends.
Blunt end endonucleases, such as Alu1, cut DNA in the middle of the recognition site. This results in a straight cut and no overhanging nucleotides.
Sticky end endonucleases, such as
EcoR1, do not cut in the middle of the recognition site, resulting in a staggered cut with overhanging, unpaired nucleotides.
called ‘sticky’ because
unpaired nucleotides will want to stick to, a complementary set of unpaired nucleotides
Ligases are
enzymes that join two fragments of DNA or RNA, acting like molecular glue.
ligase - The enzyme will
catalyse the formation of phosphodiester bonds between the two fragments to merge them together.
ligase enzyme function as
as the reverse of endonucleases. they can join together any blunt or sticky ends.
polymeras
amplify section of rna an dna, require a primer to attach to the start of a template strand of DNA.
CRISPR-Cas9:
a complex formed between gRNA and Cas9 which can cut a target sequence of DNA.
Protospacer:
a short sequence of DNA extracted from a bacteriophage by Cas1 and Cas2, which has yet to be incorporated into the CRISPR gene
how crispr works
When a bacterium is infected by a bacteriophage, there are three steps to fighting the virus with the CRISPR-Cas9 system:
Exposure
Expression
Extermination.
steps to crispr
step 1 exposure - bacteriophage injects dna into bacterium, which identifies the viral dna as foreign, cas1n2 enz cut short section of viral dna
step 2 expression - crispr spaces transcribed along with a palindrome, n converted into rna molecule known as guide rna, this binds to cas9 to create crisper cas9 complex, gRNA forms hairinloop structure
step3 extermination - crispercas9 compelx scans cell for invading bacteriophage dna, then cas9 cleaves phosphate sugar backbone to inactivate virus then cas9 contains 2 active sites to cut strands of dna blunt
What happens when the viral DNA is cut?
enzymes within the bacterium will naturally act to repair it.re[aor mechanisms can make mistaes and become useless, so process will cont till mutation occurs
Genetic modification:
manipulation of an organism’s genetic material
Gene knockout:
scientists prevent the expression of a target gene to understand its function in an organism
Gene knock-in:
scientists add specific gene into genome
cripr cas 9 in gene editing
-gene mod can amend deletrious mutations
-gene therapy splves number of health prob
-increases crop productivity and eliminats genetic diseases
Purpose of the polymerase chain reaction
(PCR) is a DNA manipulation technique that amplifies DNA by making multiple identical copies.
used by scientists
whenever there is an insufficient amount of a DNA sample for testing.
Primer:
a short, single strand of nucleic acids that acts as a starting point for polymerase enzymes to attach
Taq polymerase:
a heat-resistant DNA polymerase enzyme, which amplifies a single-stranded DNA molecule by attaching complementary nucleotides
PCR required materials
- DNA sample
-Taq polymerase is required to bind complementary nucleo to the single-stranded DNA
-Nucleotide bases must be constantly available to create a new strand that is complementary
-Sequence-specific DNA primers join to the 3’ end of single-stranded
process
- 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.
- Annealing – 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.
- Elongation – the DNA is heated again to 72 °C, Taq polymerase binds to the primer, and begins synthesising a new complementary strand of DNA.
4.repeat
The process of gel electrophoresis
laboratory technique used by scientists to measure the size of DNA fragments.
example of cripser cas
- guide rna is created that matches target dna sequence on numbat genome
- cas9 n gRNA r combined to prod crisper cas 9 complex
- numbat zygote cells r injected with the complex
4.gRNA recognises target numbat dna sequence to be removed
5.cas9 cuts bth strands of dna, removing target dna sequence
- thylacine dna is incorporated into nubat dna
reason for including sample of animals dna in irst 2 lanes
neg and pos control to see if crisper worked
process of gel elctropherisis
1. dna sapmles r placed in the walls at one end of the gel using micropipette
2.electric current is passed trough the gel using 2 electrodes, pos and neg placed at op ends,dna move from wells to pos
3.smaller dna frag move faster through gel. current off and stops moving, sep based on size
4.dna is diff to see with naked eye so gel is stained with fluorescent dye
