DNA Manipulation

Endonuclease

Cut at specific restriction sites

Sticky ends

Overhanging nucleotides at ends

Blunt ends

No overhanging nucleotides

Ligases

Join DNA/RNA together by catalysing the formation of phosphodiester bonds between nucleotides

Polymerases

Synthesise a DNA/RNA polymer from nucleotide monomers

Restriction endonuclease/restriction enzyme

Cuts DNA at a specific restriction site

Ligase

Joins DNA/RNA together by catalysing formation of phosphodiester bonds between nucleotides

Polymerase

Catalyses the extension of a DNA/RNA polymer by adding nucleotide monomers to the 3' end

CRISPR

Clustered regularly interspaced short palindromic repeats

CRISPR

Cas9

gRNA

Complementary to spacer, is transcribed and binds to Cas9 enzyme forming a CRISPR

Cas9 enzyme

Recognises the PAM sequence and cuts any DNA that is complementary to the gRNA, creating blunt ends.

NHEJ

The cell's DNA repair mechanism that attempts to repair DNA cuts, errors introduced render the virus non

Spacer

Does not have a protospacer adjacent motif, hence CRISPR

CRISPR

Cas9 complex structure

sgRNA

Synthetic gRNA, is a single RNA strand.

Gene editing with CRISPR

Cas9

Non

homologous end joining

Homology directed repair

Modified DNA with homologous ends is used for homology directed repair to insert desired gene (knock in).

Knock out

Knock out of deleterious alleles of genes.

CRISPR applications in research

Locate specific genes in the genome by attaching a fluorescent protein to Cas9 (conjugated).

Gene disruption

Disrupting expression of a gene to observe effects of the protein being knocked out

CRISPR applications in agriculture

Increase yield by either introducing pest/herbicide resistance to crops or promoting increased growth rate.

CRISPR applications in disease

Introduce genes which code for proteins that decrease susceptibility to infectious disease.

Cancer gene modification

Modify cancer

Limitations of CRISPR

Cas9

Genome alteration requirement

To alter the genome, the embryo has to be altered so that the DNA of each cell is altered.

PAM role in CRISPR

To ensure self

CRISPR definition

CRISPR stands for Clustered regularly interspaced short palindromic repeats.

CRISPR

Cas9 function

gRNA in CRISPR

Cas9

Cas9 enzyme function

Cas9 enzyme recognises the PAM sequence and cuts any DNA that is complementary to the gRNA, creating blunt ends.

DNA repair mechanism

After DNA is cut, the cell's DNA repair mechanism (NHEJ) attempts to repair, errors introduced render the virus non

Spacer DNA protection

CRISPR

CRISPR

Cas9 in gene editing

Cas9 complex recognition

Cas9 complex recognises PAM and if DNA is complementary to sgRNA, cuts the DNA.

Blunt end cut

Cas9 cuts the DNA complementary to the spacer forming a blunt end cut.

Fluorescent protein attachment

Locate specific genes in the genome by attaching a fluorescent protein to Cas9 (conjugated).

Pest/herbicide resistance

Introduce pest/herbicide resistance to crops.

Increased growth rate

Promote increased growth rate.

Disease susceptibility reduction

Introduce genes which code for proteins that decrease susceptibility to infectious disease.

Gene knock in precision

It is difficult to achieve gene knock in with precision.

Gene knock in success

Gene knock in is not consistently successful.

Embryo alteration

To alter the genome, the embryo has to be altered so that the DNA of each cell is altered.

PAM efficiency

To increase efficiency by allowing the CRISPR

Plasmid

Circular piece of self

Recombinant plasmid

Circular piece of self

Vector

Means of introducing foreign DNA into an organism.

Recombinant plasmid production

Gene of interest is isolated with introns removed either through synthetically making the DNA in a lab or by producing copy DNA using reverse transcriptase to produce a DNA sample complementary to mature mRNA.

Plasmid vector acquisition

Plasmid vector with antibiotic resistance gene and reporter gene is obtained.

Restriction endonuclease

A restriction endonuclease with the same restriction site is used to cut both the gene of interest and plasmid vector to form complementary sticky ends.

Hydrogen bond formation

The plasmid vector and the gene of interest are mixed allowing the formation of hydrogen bonds between the complementary sticky ends.

DNA ligase function

DNA ligase joins the gene of interest to the plasmid vector by catalysing the formation of phosphodiester bonds in the sugar phosphate backbone.

Universal genetic code

The genetic code is universal, meaning that the same codon will code for the same amino acid in almost all organisms.

Bacterial transformation process

Mixture of recombinant plasmids and bacteria undergo either heat shock or electroporation to make the plasma membrane more permeable, increasing the likelihood of plasmid uptake.

Antibiotic culture selection

A transformed bacteria is a bacteria which has taken up the plasmid vector will be conferred antibiotic resistance by the plasmid and hence survive on an antibiotic culture.

Reporter gene

Allows for detection of successful gene insertion.

Transformed bacteria

Bacteria that has taken up the recombinant plasmid and does not express the trait conferred by the reporter gene due to disruption by the gene of interest.

Insulin production

Process involving recombinant plasmids to produce insulin subunits.

Plasmid vectors

Contain the ampR gene and tetR gene, used for insulin production.

EcoRI and BamHI

Restriction enzymes used to cut the insulin A or B subunit gene and the plasmid for correct orientation.

Triplet coding for methionine

Added to the beginning of the insulin gene during the plasmid preparation.

DNA ligase

Enzyme that ligates the insulin subunit gene and the plasmid.

Ampicillin

Antibiotic used to eliminate untransformed bacteria.

Tetracycline

Antibiotic used to culture colonies, where those that die contain the recombinant plasmid.

LacZ

Gene inserted into plasmids to produce a fusion protein that protects the insulin subunit gene.

X

gal

Heat shock

Adding calcium ions and changing temperature rapidly to create adhesion zones in bacteria for plasmid entry.

Electroporation

Applying short high voltage pulses for plasmids to overcome the barrier of the cell membrane.

Gene of interest isolation

Process of isolating a gene with introns removed either through synthetic DNA or reverse transcriptase.

Hydrogen bonds

Formed between complementary sticky ends of the plasmid vector and the gene of interest.

Phosphodiester bonds

Formed in the sugar phosphate backbone by DNA ligase to join the gene of interest to the plasmid vector.

Bacterial Transformation

The process where bacteria take up recombinant plasmids through methods such as heat shock or electroporation.

Recombinant Plasmids

Plasmids that have been genetically modified to contain genes of interest, such as insulin subunit genes.

Antibiotic Selection

The use of antibiotic

Untransformed Bacteria

Bacteria that have not taken up the plasmid and therefore lack antibiotic resistance, resulting in their death on antibiotic cultures.

AmpR Gene

A gene that confers resistance to the antibiotic ampicillin, used in plasmid vectors.

TetR Gene

A gene that confers resistance to the antibiotic tetracycline, used in plasmid vectors.

EcoRI

A restriction enzyme used to cut DNA at specific sequences, facilitating the insertion of genes into plasmids.

BamHI

A restriction enzyme that cuts DNA to create complementary sticky ends for gene insertion.

LacZ Gene

A gene that codes for beta

Fusion Protein

A protein created by ligating the insulin subunit gene with LacZ, which protects the insulin gene from degradation.

Disulfide Bonds

Chemical bonds that form between cysteine residues in proteins, essential for the formation of functional human insulin.

Colony Inspection

The process of examining bacterial colonies to determine which have successfully taken up the recombinant plasmid based on phenotype expression.

Protein Extraction

The process of isolating proteins from transformed bacteria after they have been cultured.

Purification

The process of isolating a specific protein, such as insulin, from other cellular components after extraction.

PCR

Polymerase chain reaction

Denaturation

Sample is heated to 94°C to break the hydrogen bonds between complementary base pairs, denaturing the DNA sample, forming single

Annealing

Single

Elongation/extension

DNA is heated to 72°C, taq polymerase synthesises new complementary strand of DNA by adding nucleotides to the 3' end.

Role of primers in PCR

Primers 'bracket the gene of interest', ensuring only it is amplified. Primers provide a binding site for Taq polymerase to extend from.

Components of PCR

Thermocycler, Forward and reverse primers, Taq polymerase, DNA sample, Free

Purpose of PCR

PCR is used to amplify the amount of DNA present, so there is sufficient DNA to be detected during analysis by the human eye/other equipment.

Primer

Single stranded pieces of nucleic acids that are complementary to target DNA. They act as a starting point for polymerase enzymes to attach to.

Buffer solution

Prevents pH of solution from changing during PCR.

Gel electrophoresis

Separates DNA samples by length.

Process of gel electrophoresis

DNA samples are loaded into wells in an agarose gel. Gel is immersed in buffer solution to help carry current. Current is supplied. DNA migrates to the positive electrode. Larger DNA samples face more resistance from the gel, so move more slowly. DNA fragments are separated into bands. Current is turned off. Bands are dyed with ethidium bromide to enhance visibility.

Standard ladder

A mixture containing DNA fragments of known lengths, from which, by comparison, the length of unknown fragments can be estimated.

Property of DNA for gel electrophoresis

It is negatively charged. Agarose gel resistance makes different sizes of DNA fragments travel different distances.

Factors affecting DNA travel distance

Voltage (positive correlation), Gel density (negative correlation), Buffer concentration (more ions = positive correlation), Time (positive correlation).