DNA Manipulation

Year 12 Biology U3 AOS 1

Enzymes that manipulate DNA

• Endonucleases → cuts DNA (scissors)

• Ligases → joins DNA (glue)

• Polymerases → amplify DNA (multiply)

Endonucleases

enzymes that break bonds between nucleotides of nucleic acids

Sticky ends

staggered cuts through a double-stranded DNA, resulting in overhanging nucleotides

• Called “sticky” as unpaired nucleotides will be attracted to a complementary set of unpaired nucleotides

• Ensures an inserted gene is oriented correctly

Blunt ends

straight cuts through a double-stranded DNA, resulting in no overhanging nucleotides

Restriction enzymes

a type of endonuclease that are produced by bacteria + cut DNA at specific recognition sites

Restriction enzymes vs. Cas9

• Restriction enzymes — only act on specific restriction site which cannot be programmed

• Cas9 — versatile so it can be programmed to target ANY specific sequence dictated by a piece of gRNA

Ligases

enzymes that join nucleic acid fragments together by creating bonds between a sugar and a phosphate (backbone)

• DNA ligase → joins two DNA fragments

• RNA ligase → joins two RNA fragments

Polymerase

enzyme that synthesises a polymer from monomers, such as forming a DNA strand from nucleic acids

• Requires a primer → a short, single strand of nucleic acids that acts as a starting point for polymerase enzymes to attach

CRISPR-CAS9

a complex formed between gRNA and Cas9 which can cut a target sequence of DNA

• Bacteria uses this complex for protection from viruses

• Scientists modify this complex to edit genomes

Cas9

an endonuclease that can be programmed to cut any specific DNA recognition sites

sgRNA (single-guide RNA)

guides Cas9 and tells it where to cut.

• Made up of ribose sugar, phosphate and nitrogenous bases

• Changes in sgRNA determines how Cas9 is programmed

• Scientists can create sgRNA with any sequence to cut at any known target gene

Effect of temperature on Cas9 reaction rate

• Reaction rate is greatest at optimum temperature

• Above opt. temp. → enzyme denatures → decreasing rate of reaction

• Below opt. temp. → less kinetic energy (fewer collisions) → slow rate of reaction

FIRST step of CRISPR-Cas9 in bacteria

Exposure

• Bacteriophage (virus) injects its DNA into a bacterium + recognises the viral DNA as foreign

• Cas1 and Cas2 cut a short section of viral DNA (protospacer) as it matches the PAM

• Stored in the bacterium’s CRISPR array as a spacer (+ bacterium now has a genetic “memory” of the viral DNA)

SECOND step of CRISPR-Cas9 in bacteria

Expression

• When virus attacks again, CRISPR array is transcribed into guide RNA (gRNA)

• gRNA binds to Cas9 to create a CRISPR-Cas9 complex

• It is directed to the viral DNA complementary to gRNA sequence

THIRD step of CRISPR-Cas9 in bacteria

Extermination

• CRISPR-Cas9 complex scans the call for viral DNA complementary to gRNA sequence

• Upon finding target, Cas9 cleaves viral DNA by making a double-strand break in the sugar-phosphate backbone to inactivate the virus

• After successful cleavage, new viral sequences are added to CRISPR array to protect bacterium from future attacks by the same virus

CRISPR-Cas9 in Gene Editing — Process

1. Identify the sequence of the gene to be targets

2. Create sgRNA complementary to the target gene

3. Combine sgRNA with Cas9 and introduce into organism

4. sgRNA + Cas9 looks for PAM sequence on DNA strand

5. When PAM sequence is found, Cas9 looks for target gene sequence

6. DNA is unwinded to let Cas9 cut the target gene

7. DNA sequence is altered (by additional processes) to either switch off or repair/alter the gene

Gene Knocks IN Gene Editing

a new DNA sequence is inserted into the DNA break

• Allows a faulty gene sequence to be replaced with the correct one to restore gene function

Gene Knocks OUT Gene Silencing

errors (mutations) can occur as the cell’s normal repair mechanisms mend the broken DNA, causing the insertion or deletion of bases

• Changes the way the nucleotide sequence is read

• Disables gene function + produces a STOP signal

• SILENCES the gene

PAM (Protospacer Adjacent Motif)

a very short segment (2-6) of nucleotides (NGG) on the DNA that provides a binding site for Cas9

• Without PAM, Cas9 will not cut DNA even if a sequence complementary to the gRNA is present

• Makes CRISPR more efficient

Role of PAM

Protective of bacteria

• Bacteria never have a PAM sequence in their own DNA

• - Ensures Cas9 cannot cut the bacterium’s own DNA

PCR (Polymerase Chain Reaction)

PCR amplifies a specific DNA sequence by making multiple identical copies

• Used by scientists when there is insufficient DNA samples for testing

• Entire genomes are not copied, but certain genes are, to make process more efficient

• After each PCR cycle, amount of DNA is DOUBLED

Purpose of PCR

amplifies a sample of DNA to increase the quantity of DNA available

FIRST step of PCR

Denaturation

• DNA is heated to ~90-95℃ to break the hydrogen bonds between the bases and separate the strands, forming single-stranded DNA

SECOND step of PCR

Annealing

• The single-stranded DNA is cooled to ~50-55℃ to allow the primer to bind to complementary sequences on the single-stranded DNA

THIRD step of PCR

Elongation

• The DNA is heated again to ~72℃, the optimal temperature for DNA polymerase to synthesise new DNA strands by adding nucleotides in 5’ to 3’ direction

FOURTH step of PCR

Repeat

• The cycles (steps 1-3) is repeated multiple times to create more copies of DNA

Forward Primers

binds to the start codon at the 3’ end of the template strand

• Causes Taq polymerase to synthesise a new DNA strand in the same direction that RNA polymerase would function

Reverse Primers

binds to the stop codon at the 3’ end of the coding strand

• Causes Taq polymerase to synthesise a new DNA strand in the reverse direction that RNA polymerase would function

Importance of Primers

• Having two primers is necessary as the 5’ ends of both the template + coding strands are different

• As Taq polymerase only functions towards the 3’ end, a primer is needed for both strands to facilitate directionality

Taq polymerase vs. DNA polymerase

• Taq → very high optimal temperature, working optimally at 72℃

• DNA → would denature, being incapable of synthesising a new strand at that temperature

Gel Electrophoresis

a technique used to compare DNA samples by separating DNA fragments according to their size

• DNA samples are often amplified using PCR + cut using restriction enzymes BEFORE being separated

• Gel is made of agarose, immersed in a buffer solution

• DNA is stained with ethidium bromide to see more clearly under UV light

• An electric current is used to separate the fragments

Wells

• DNA samples are placed in wells in the gel

• These wells are always placed at the negative electrode as DNA has a negative charge

• Usually the first well contains a sample with DNA fragments of a known size (DNA ladder)

Gel Electrophoresis — Process

• When the power is turned ON → the DNA moves through the gel towards the positive electrode

• Because DNA has an overall negative charge

• Smaller DNA fragments move more quickly + travel further through the gel

STR (Short Tandem Repeats)

short non-coding DNA segments that vary in length between individuals making them very useful for DNA fingerprinting

Bacterial Transformation

when scientists insert new genes into bacteria + turn them into a protein-producing factories

gene of interest

the gene we want the bacteria to express (e.g insulin gene)

plasmid vector

a circular ring of DNA used to transport the G.O.I into the bacteria

Recombinant plasmid

plasmid that has the G.O.I inserted into it (basically G.O.I + P.V)

Restriction endonucleases

cuts G.O.I + P.V, producing complementary “sticky ends” that allow the gene to be inserted into the plasmid

DNA ligase

joins G.O.I + P.V together by forming phosphodiester bonds between each DNA sugar-phosphate backbone

Creating a Recombinant Plasmid — Process

1. The gene of interest is removed from the DNA strand using restriction enzymes

2. The plasmid is also cut using the SAME restriction enzyme

3. G.O.I + plasmid have complementary sticky ends + easier to join

4. G.O.I is positioned in plasmid → ligase joins the two pieces of DNA

(Steps 1-3) Bacterial Transformation — Process

1. The recombinant plasmid combines with bacterial cells

2. Mixture is treated with heat/electricity shock to encourage bacteria to take up the recombinant plasmid

3. Bacteria that take up the recombinant plasmid are transformed

FOURTH step of Bacterial Transformation — Process

4. Not all bacteria will take up recombinant plasmid

• Only the bacteria with antibiotic resistance gene (transformed) will survive

• Allows us to only culture the transformed bacteria that will express G.O.I

FIFTH step of Bacterial Transformation — Process

5. Colonies of transformed bacteria should be observed on nutrient agar

• Transformed → should form colonies as they contain tcl gene, conferring resistance to tetracycline in the culture

• Untransformed → unable to form colonies as they do not contain tcl gene and die

EXAMPLE WITH E.COLI → PRAC DONE IN CLASS!

Human Insulin

has a quaternary structure, made of 2 polypeptide chains

• Two separate plasmids must be produced + placed in separate bacteria → one for Chain A and Chain B

• When the polypeptides are extracted, they are combined to form functional human insulin

Reporter Genes

used in bacterial transformation to indicate whether the G.O.I is being expressed or not

• Code for proteins that are easily visualised

• Allows researchers to select for bacteria that have taken up the recombinant plasmid (will express G.O.I) → similar to antibiotic resistance gene (with/without G.O.I)

Examples of Reporter Genes

• GFP → causes bacteria to glow green

• Lac z → causes bacteria to turn blue

GMO — Genetically Modified Organism

an organism that has had its DNA artificially altered in any way.

Examples of GMOs

• Inserting DNA from SAME species

• Removing DNA

• Silencing genes

• Replacing nucleotides

TGO — Transgenic Organism

an organism that has had a gene from a different species inserted into its genome

Example of TGO

• Inserting DNA from ANOTHER species

Non-GMO

an organism that genetic material has not been altered using genetic engineering or other modern biotechnology techniques

Examples of Non-GMO

• Selective Breeding

• Vaccination