4.5 APPLICATION OF REPRODUCTION AND GENETICS 2

PCR useful

• crime scene analysis

• paternity testing

• disease diagnosis

gel electrophoresis

• restriction endonuclease Dna sample cut to specific nucleotide sequecnes by

• Fragments loaded into wells one end of agarose gel

• Voltage applies across gel

• Fragments separates by length

• Smaller migrate easier through pores travel further

• Through covered in nylon membranes which picks up DNA fragments southerns blotting

• DNA probes attach to specific part of fragments

• Xray film places on blot and exposed to give DNA fingerprint

DNA ladder fragments of known length created at the same time to estimate length of fragments under tets

PCR

1. Denature

• DNA heated to 95°C

• Hydrogen bonds between bases break

• Double-stranded DNA separates into single strands

2. Annealing 50°C

• Primers bind to complementary DNA sequences

• mark starting point for DNA synthesis

3. Extension 70°C

• Taq DNA polymerase adds complementary nucleotides

• Forms phosphodiester bonds

• New DNA strands produced

4. Repitition

• Cycle repeats many times (~40 cycles)

• DNA doubles each cycle

• Produces billions of DNA copies

Taq polymerase

• Thermostable enzyme from bacteria Does not denature at high temperatures

• Needed because PCR repeatedly heats DNA

Primers

• Short single-stranded DNA sequences

• Complementary to target DNA

• Allow DNA polymerase to begin replication

WHy is P

Transfer genes into uses

• Bacteria to useful products (insuling)

• Plants and animals - acquire new charectierstics

• Humans -reduce effects of genetic diseases

Recombinant DNA fromed when

Genetic material from 2 species combines

Transgenic

• Organism DNA from another species

• Introduced DNA is donor into host

Restriction endonuclease enzymes

Cut DNA

DNA ligase

• enzyme

• reform sugar phosphate backbone

• formation of sticky ends

What cDNA overcomes

• Locating gene

• No introns present

• Avoids gene being cut into fragments

• No post-transcriptional processing needed

• Easier for bacteria to express gene

Restriction enzyme method of preparing DNA

• Human DNA extracted

• Restriction endonuclease enzymes cut DNA at specific recognition sites

• Desired gene is cut out of chromosome

• Gene can be inserted into plasmid

mRNA and cRNA method of DNA

• mRAN extracted from cells actively producing required protein (insulin)

• Reverse transcriptase make complementary DNA (cDNA) from mRNA

• DNA polymerase produces second DNA

• Double stranded cDNA containing gene formed

mRNA / cDNA Method of Producing Recombinant DNA

• mRNA is extracted from cells actively producing the required protein (e.g. insulin).

• Reverse transcriptase is used to make complementary DNA (cDNA) from the mRNA.

• DNA polymerase produces the second DNA strand.

• A double-stranded cDNA containing the required gene is formed.

Inserting the gene into a plasmid

• A plasmid is cut open using the same restriction enzyme used to isolate the gene.

• This creates complementary sticky ends on both the gene and plasmid.

• The gene is mixed with the plasmid, allowing sticky ends to pair together.

• DNA ligase joins the sugar-phosphate backbone, permanently joining the gene to the plasmid.

• The plasmid now contains recombinant DNA.

Putting recombinant DNA into bacteria

• Calcium ions encourage bacteria to take up the recombinant plasmids.

• The bacteria are cloned in fermenters to produce large amounts of the required protein.

To make a good vector

• Self replicating and small

• Not be broken down by host enzymes

• Not stimulate immune response

• Be able to be screened and confirm gene actually inserted into vector

• Have markers to allow host cells that have succesfully taken vector to be identified

Whats marker genes

• Plasmid inserted into bacteria

• Bacteria grown on antibiotic medium

• Only bacteria containing plasmid survive

• Allows scientists to identify transformed bacteria

purpose

• Identify recombinant bacteria

• Select bacteria containing plasmids

Pros genetic engineering

• Medical products (insulin)

• Prevention and treatment of disease

• Enhancing crop growth - killing plant pests

• Environmental use - detecting envrionmental hazards

Cons of genetic engineering

• Plasmids easily transferred (antibiotic resistance)

• Fragments of human DNA used to make samples may contain oncogenes or gene activate proto-oncogenes

• Microorganisms with new gene may become threat if released

• Newly introduced gene may disrupt the normal fucntion of other genes in ways not yet under stood

Tomatoes GM

Gene inserted for slow ripening

• Better keeping quality

• long shelf life

• Reduced food waste during transport

GM soya beans

Gene inserted for herbicide resistance

• crops survive herbicide spaying

• Weeds killed without damaging crop

• Higher yields

• Easy weed control

Creating insect resistant tomato plant

1. Cut gene restriction endonucleases

2. Insert in vector with antibiotic resistance marker gene

3. Copy vector in bacteria

4. Coat tungsten with DNA vectors

5. Load vector coated particles on teflon bullet

6. Load bullet into gene gun

7. Shoot gene gun released particles high velocity penetrate plant cells

8. Vector enters cells genes incorporated into plant genome

9. Cells plated selective antibiotic media

10. Cells transferred to medium containing plant grwoth

Benefits and disadvantages of gm

Good

• Superior nutrition and keeping quality

• Higher yield

• reduction in pesticide use(engineered for fungal pathogens and insect attack)

Bad

• Dispersal of pollen from crops engineered for herbicide resistance to wild relatives

• Unknown effect eating new portien in crop

• Reduction in biodiversity

Aim of gene therapy

Treat genetic disease by replacing defective allles in a patient with copies of new and correct DNA sequence

Gene therapy

• Replace defective alleles

• Restore production of normal proteins

• Reduce symptoms of genetic disorders

Somatic gene therapy

Repeated reguarly treated cells worn out

• genes inserted

• Only affects treated person

• Not inherited

Germ line therapy

• Genes inserted into gametes / embryos

• Can be inherited by offspring

• Controversial genes interact with each other

Duchenne muscular dystrophy

Sex linked genetic recessive 1/3400

• mutation in dystrophin gene → failure to produce

• Important structural component of muscle tissue

• Severe wasting of muscle

DMD symptoms

• Muscle weakness

• Wheel cair

• Breathing difficulties

Exon skipping

Drisapersen treat DMD introduce molecular patch pver exon

• Mutation make the gene readable again

• Remaining axons join ribosome can read mRNA

• Reduce severity of symptoms

Genomics

Study of structure function and evolution and mapping genomes

Examples

• Human genome

• 100k project

Genomics aims

• More accurate diagnosis

• Better prediction of effects of drugs

• Improved design of drugs

• New improved treatments for diseas

Next generation sequencing

• Sequence entire genome in few hours faster than sanger

• Cheaper

Tailoring therapies to individual patients to have unique treatment

Sanger and ngs comparison

SS

• Slow

• Small DNA sections

• Expensive for large genomes

NGS

• very fast

• whole genome

• More cost effective

What is tissue engineering

Artificial production of tissues or organs using stem cells

• Grown in culture

• divide by mitosis

• differentiate into specialised

• Tiss e/ organ formed

WHy stem cells useful

Unspecialised

Develop to many types

Types of stem cells

• Totipotent cells - from every cell type in organism

• Embryonic stem found in 3 day embryo pluripotent form every type of cell in the body easily grow large num

• Some adult tissue - bone marrow, brain has some, muscle replace cells lost through normal wear and tear injury disease - limited

Ethicals embryonic stem

• Source of embryonic - against principle of life

• Moral status - moral rights

All enzymes in genetic engineering

Restriction endonuclease cut out the desired gene

Same endonuclease to cut open plasmids

Producing complementary sticky ends

Ligase join gene to plamsid

Advantage of resistance to herbicide (weed killer)

Not kill crop but kills weeds

Reducing competition in the field

Increase yield

Objections against resistance to hericide

Increased use o fherbicide

Reduction in biodiversity

Dispersal of pollen from croped for herbicide resistance to wild relatives

Unknown effects of eating new protein

What is meant by restriction endonuclease

Cuts DNA at a specific base sequence

CRISPR techniqe to make fertile mosquito

• Make guide RNA with complementary sequence of targe / fertility gne

• Insert CRSPR in to egg / cell

• Incubate to grow

• Remove gene that makes fertile

Benefit of sterilised mosquito and ehtical

• Reduced fertility

• Less mosquitos = less malaria

• Unknown effects

• Food chain biodiversiity

• Right of humans to make species extinct

Gentically engineering mRNA - cDNA

• Exctract mRNA from bacterium

• Reverse transcriptase to synthesise cDNA strand using mRNA as template

• DNA polymerase produce double stranded DNA

• Sticky ends added to end of each DNA strand

• DNA copies using PCR

After cDNA

• Plasmids from bacteria isolated

• Restriction endonuclease open plasmid at specific point

• Sticky ends complementary to DNA

• DNA ligase bind DNA to plasmid

• Antibiotic resistant genes select bacteria containing recombinant plasmids

• Describe how cDNA fragment contain gene be prepared and process by genetically engineered plasmids

• Explain concerns over techniques to increase resistance to insect pests in crops

Prepare

• Exctract mRNA from bacterium

• Reverse transcriptase to synthesise cDNA strand using mRNA as template

• DNA polymerase produce double stranded DNA

• Sticky ends added to end of each DNA strand

• DNA copies using PCR

• Plasmids from bacteria isolated

• Restriction endonuclease open plasmid at specific point

• Sticky ends complementary to DNA

• DNA ligase bind DNA to plasmid

• Antibiotic resistant genes select bacteria containing recombinant plasmids

Concers

• Dispersal of pollen from engineered crops to wild

• Unknown effects of eating new protien which is toxic to insects

• Reduction in biodiversity

• Could result to resistance to protein in insects

Problems of virus as vector to tread DMD

• Cause immune response against virus

• Problems introducing gene to muscles wont reach it

• Virus may destroy cells become pathogenic

• May affect other genes (oncogenes)

Advantage of a drug to remove mutated DMD rather than patch

• don’t have to repeat

• more permanent treatment

• Can pass on to next generation

How have Human genome project made artificial genes possible

The sequence of DNA bases is known

Advantages of artificially synthesised gene rather than extracting

• Dont need to locate the gebe / isolate

• No restriction endonuclease enzymes

• No finding functional mRNA

• Extracting gene may damage

• No introns only exons

2 Enzymes needed for successful insertion of a gene in plasmid

• Restriction endonuclease cut DNA in plasmid at specific point

• DNA ligase binds the plasmid /vector to Gene

For plating recombinant DNA why use antibiotic agar plate first

• Only bacteria which have a plasmid can survive

• shows that these bacteria have taken up a plasmid

When X is inserted to ampillicin gene (plasmid also contains tetra resistance ) explain 2 plates

Tetra

• Only bacteria with the plasmid survive

Ampillicin

• Gaps in colonies Died

• Not resistant to ampilicin

• taken up X gene → Recombinant 🥳 DNA

• Ampicillin gene not expressed

After replica pad why is there missing colonies

• Missing died did not grow are bacteria not resistant to new plate

• Because theyve taken ip recombinant plasmid

• So theyr not expressed

Essential property of inserted gene that will need to be confirmed before third plate

• Will produce gene when in culture and is expresed

Explain gene therapy and one of the techniques involved and example

• Insertion of functional gene into cells replace faulty gene

• Germ line functional gene added to embryo + inherited

• Somatic gene therapy functional gene placed in body cells

• Not permanent

• Cystic fibrosis CFTR gene inhaled

• Restriction endonuclease ligase vectors

Aims of human genome project

Sequence identify position of genes in human genome

• Understand genetic disorders improve diagnosis and treatment

Use in gene therapy DMD

• DMD caused by mutation in dystrophin gene

• Failure to produce dystrophin → muscle weakness

• Exon skipping introduce molecular patch over exon with mutation

How was hgp made gene editing technique possible

• Identify position of all genes

• Know base sequence of normal gene and RNA guide

Why 2 different primers in pcr

different nucleotide sequence at each end/one primer for each strand of DNA

Why is it important to use primers which are specific to a certain gene on each chromosome

Enable specific gene to be copies

Why express quantitiy of genes as a ration

• Quantity doesn’t matter

• Ratio will be the same