Gene Technology: Plasmids and PCR

Gene Technology (Parts 1-4)

Advantages of Using Plasmids

• Small and circular piece of double-stranded DNA.
• Replicates independently with multiple origins of replication.
• High copy number.
• Easy to extract from bacteria.
• Can be cut using restriction enzymes or endonucleases.
• Allows gene or DNA insertion.
• Can be taken up by bacteria.
• May contain genes for antibiotic resistance or carry marker genes.
• Helps in identifying transformed bacteria.
• Acts as a vector.
• May carry a promoter.

Polymerase Chain Reaction (PCR)

• Produces a large number of copies of a length of DNA or amplifies DNA.
• Rapid and automated process.
• Requires only a small DNA sample.
• DNA is denatured or separated into two strands by heat at 95°C.
• DNA primers are added.
• Annealing occurs at 60-65°C, involving complementary base pairing.
• DNA or Taq polymerase replicates the template strand at 70-75°C.
• The process is repeated after heating again to separate strands.
• Taq polymerase is heat stable with a high optimum temperature and doesn't need replacing each cycle, making the process efficient.
• Taq polymerase comes from Thermus aquaticus, a bacterium living in hot springs.

Bioinformatics

• Analysis of biological data using computer software.
• Includes databases of gene, DNA, protein, and amino acid sequences.
• Large databases allow fast, accurate, and efficient data sharing and pooling.
• Can predict amino acid sequences or protein structure from DNA sequence data.
• Used for comparisons between DNA sequences.
• Aids in finding methods to control parasites, e.g., in vaccine development.
• Facilitates personalized medicine.
• Helps identify new diseases and discover common ancestors.
• Used in phylogenetic analysis and for biodiversity and new species studies.

Electrophoresis

• VNTR (variable number tandem repeat) sequences are referenced.
• DNA quantity is increased by PCR.
• DNA is fragmented by restriction enzymes or endonucleases.
• Fragments are loaded into wells in an agarose gel at the negative or cathode end.
• A buffer or electrolyte is used.
• Direct current is applied.
• Phosphate groups in DNA give it a negative charge.
• Negatively charged DNA is attracted to the anode or positive electrode.
• Shorter pieces with smaller mass move further and faster.
• Pieces are transferred to a membrane, nylon, or nitrocellulose via Southern blotting.
• The membrane is heated to separate strands.
• Probes or fluorescent dyes are added.
• Bands are visualized using X-ray film, UV light, or lasers.

Microarray (for Gene Expression Analysis)

• Probes are single-stranded DNA (ssDNA).
• Each probe is unique to a particular gene.
• Probes correspond to thousands of different genes.
• mRNA is extracted and used as a template to make cDNA via reverse transcriptase.
• cDNA is linked to fluorescent dye.
• cDNA is added to the microarray.
• cDNA binds to or hybridizes with probes by complementary base pairing.
• Excess cDNA is washed off.
• When exposed to UV light, fluorescence indicates expressed genes.
• Fluorescence intensity shows the level of gene expression.

Advantages of Obtaining Insulin Using Genetic Engineering

• More effective because it's identical to human insulin.
• Provides a more rapid response.
• No immune response is triggered.
• Highly pure, eliminating the risk of infection transfer.
• Cheaper for larger volumes.
• Has fewer ethical and moral objections because animals aren't involved.
• Suitable for people who developed tolerance to animal insulin.

Promoter

• Initiates gene transcription, switches on the gene, or causes gene expression.
• Allows RNA polymerase and transcription factors to bind.
• Enables RNA polymerase to recognize which DNA strand will serve as the template for transcription.
• Supports RNA polymerase binding to transcribe the gene of interest.
• Determines the level of gene expression at the right time and in sufficient amounts.
• Gene insertion can be difficult if an existing promoter is nearby and risks disrupting expression of another gene.
• If inserted into any chromosome, the gene may be included within introns.
• A gene might share a promoter with a host gene that isn't switched on in that host cell.

Obtaining and Inserting the Human Insulin Gene into a Plasmid

• mRNA is obtained from beta cells of the islets of Langerhans in the pancreas.
• Reverse transcriptase makes single-stranded cDNA.
• DNA polymerase makes the cDNA double-stranded.
• Sticky ends are created.
• Plasmids are obtained and cut with restriction endonucleases or enzymes to create complementary sticky ends.
• cDNA or the insulin gene is mixed with the plasmid.
• DNA ligase seals nicks in the sugar-phosphate backbone.
• Recombinant plasmids are mixed with bacteria.
• Some bacteria take up plasmids and become transformed through heat shock, calcium chloride solution, calcium ions, or electroporation.

Genetic Screening

Preimplantation Genetic Diagnosis (PGD)

• Tested on embryos before implantation in the uterus.
• Couples can use PGD based on IVF.
• A single cell is removed from each embryo after a few cell divisions for embryo biopsy.
• The genetic makeup of the cell is checked.
• Only problem-free embryos are selected and placed in mother's uterus to implant and grow.
• This eliminates the faulty allele from the gene pool.

Advantages of PGD:

• Allows couples who would otherwise not have children to do so.
• Avoids having offspring with genetic disorders.
• Avoids late abortions.

Disadvantages of PGD (ethical concerns):

• Embryos may be destroyed if not pre-selected for implantation.
• Could lead to selection based on gender or specific traits, creating designer babies.
• May conflict with some religious beliefs.
• Genetic diseases may not develop.
• There is a risk of false positive or negative results, making the testing inaccurate.
• A healthy child might be aborted following a false positive result.
• Embryos could be damaged.

Prenatal Screening

• Amniocentesis involves obtaining a sample of amniotic fluid from a pregnant woman.
• Most samples are taken to look for chromosomal mutations.
• A disadvantage is risk of miscarriage.
• Chorionic Villus Sampling (CVS) involves obtaining a sample from the part of the placenta called the chorion.
• CVS allows parents to get an earlier warning of genetic abnormalities compared to amniocentesis.
• A disadvantage is risk of miscarriage.
• Non-invasive prenatal genetic screening analyzes fragments of DNA of cells from the placenta that have the same genotype as the fetus, and that make their way into maternal circulation.
• These samples of fetal DNA are extracted from maternal blood.

Advantages of Non-Invasive Screening:

• Reduces worry with negative results.
• Allows early treatment with positive results.
• Allows parents to consider terminating the pregnancy.
• Treatment can begin early.
• Parents can prepare for the cost of the treatment.
• Allows a child make an informed decision around having children when older.
• Allows for future planning.
• Allows couples to have children who wouldn't otherwise to due to the risk of genetic disease.
• Allows couples who are carriers to make informed decisions about starting a family, or seeking IVF and embryo biopsy.

Disadvantages:

• Spontaneous abortion.

Newborn Screening/Adult Screening

• This is analysis of a person's DNA to check for the presence of a particular allele or more alleles that are associated with the disease.
• DNA is obtained from tissue samples.
• Can be carried out on embryo, fetus, newborn or adults.
• Examples include breast cancer, hemophilia, sickle cell anemia, cystic fibrosis (CF), and Down syndrome.

Advantages:

• Can identify carriers of genetic conditions.
• Allows carriers to choose not to have children.
• Helps provide early diagnoses for diseases, such as Huntington's disease.
• Allows future planning.
• Preventative treatment may be cheaper than treating the disease itself.
• Genetic screening removes anxiety if tests are negative.

Disadvantages:

• Testing is expensive.
• Many mutations are still unknown, so may still not lead to diagnosis.
• If a person tests positive there is anxiety.
• For some diseases, no treatment is possible.
• May experience social, or financial discrimination.
• May still not develop if mutation is present.
• Couples may decide not to have children.

Gene Therapy

Complications:

• Gene therapy can be temporary.
• Treats only recessive conditions.
• Difficult to direct vectors to express the allele in specific cells.
• Small packaging capacity.
• Low probability of integration into the host genome.
• Can cause DNA mutation; gene insertion can disrupt the function of another gene.
• May be removed by the immune system before reaching target cells.
• The virus may trigger an immune response that destroys the infected cells.
• The inserted allele may be inactivated

Retrovirus

• Can insert viral DNA/healthy allele randomly into host DNA.
• Inserted allele may be inactivated.
• Inserted allele may inactivate another host gene.
• Virus may not enter target cell.
• May cause cancer, side effects, allergic response, or infection.

Solution

• The use of AAV doesn't insert into the host's genome or use lentivirus.
• Random insertion of genes into the host's genome.
• May insert its genes within another gene or within regulatory sequence.
• May activate oncogenes or switch off tumor suppressor genes, causing cancer.
• May have uncontrolled viral replication.

Steps:

• Isolate/obtain functional/normal allele.
• Insert the allele into a virus (vector).
• Remove stem/target cells.
• Insert allele gene and virus into stem cells and target cells.
• Return stem cells/T-lymphocytes/ target cells to the body.
• Obtain normal cDNA from mRNA using reverse transcriptase OR obtain normal DNA. From cells of healthy person. OR synthesize normal DNA from a gene library.
• Use probe/electrophoresis/sequencing for identification.
• PCR to amplify gene/DNA.
• Restriction enzyme/endonuclease and roles.
• DNA ligase is used to link the sugar phosphate backbone of the vector genetic material with the gene to form recombinant DNA. Then, add promoter.

Severe Combined Immunodeficiency (SCID)

• Immune cells are unable to function properly -> B and T cells.
• Unable to make adenosine deaminase (ADA) to faulty allele coding for the enzyme -> X-linked recessive allele -> Highly susceptible to infections.

Solution 1:

• • Isolate/obtain funtional/ normal. ADA allele.
• -Insert allele into virus vector.
• -Remove T-lymphocytes.
• -Insert, allele, gene, virus, into T-lymphocytes retunr T-lymphocytes , to body.

Solution 2:

Same as step 1 except , remove stem cells instead.

• Complications of this method is that the patient may have leukemia because of the random insertion of the retroviral gene into the stem cell.
• Instead bone marrow transplant will be effective.
• But it could be limited due to difficulty in finding a donor for the same marrow transplant.

Cystic Fibrosis

• Results from a faulty, autosomal recessive allele of the CFTR gene.
• There is a base deletion with CFTR.
• Or sometimes is based subsitution with (Stop Codon) incomplete CFTR.
• No functional channels for CI-ions
• CI -Ions do not move out.
• Less water leaves cell.
• Formulation of thick , sticky mucus on cell surface, can not be removed by cilia.

Complications:

• Sticky sticky mucus in lungs
• Traps bacteria more infections
• Difficulty in breathing.
• Coughing.
• Blocked pancreatic leads to diabetes.
• Block sperm Ducts/Ducts - reduced fertility.

Leber Congenital Amaurosis (LCA)

• The dominant RePE65 allele codes for a protein which regenerates the visual pigments in rods and cone cells in renta after they have been exposed to light.
• Need one allele per cell.
• Cure of disease or function of restore vision.
• Eye is a great organ as: it is small easy to target
• There is a little activity of the immune cells inside the eye , so the risk of harmful immune response to vector.

Gene Editing

CRISPR-Cas9

• A form of genetic engineering in which the genome of an organism can be changed by deleting, inserting, or replacing a length of DNA using a method
• In a lab, scientists create a strand of guide RNA that matches the base sequence needing to be edited.
• Cas9 is added to the gRNA, forming a complex
• This complex attaches to a matching genomic DNA sequence (complementary base sequence to the gRNA).
• The Cas9-RNA complex cuts both strands of the DNA and removes a section.
• This includes the insertion of one or more nucleotides to silence the, Inserting a correct faulty allele, and a deletion for frameshift.
• Deactivating the unwanted gene-no protein is synthesized.

Advantages:

• More precise technique: the gRNA can have more base pairs to allow precise cuts

• High success rater

• Can treat dominant diseases

• It is expensive

• Not everyone has access.

gRNA Synthesis

• DNA sequencing of target gene.
• Assemble gRNA template to be complimentary, to DNA sequence.

Guide RNA (gRNA)

• A single stranded RNA with specific nucleotide sequence, complimentary to specific DNA sequence of target gene.
• Forms complex with case9 endonuclease enzyme.
• Bass pair with the specific DNA sequence by hydrogen bond.
• Allows case9 enzyme to identify precise location for cutting a DNA.

RNA interference (RNAi)

• A form of post-transcriptional modification.
• A process in which RNA are involved in sequencing suppression of gene expression.
• It targets mRNA to bind to it and prevent it from translation.
• Small molecules of RNA are taken up by cells and combine with the mRNA of a gene.
• Slow down genes
• Non Peremanently
• Very Low- Level of protein

Huntington's Disease:

• Mutation,allele,gene,on chromosome 4, dominant.
• Normal recessive allele had 10-35 repeats of CAG larger number of repeat gives earlier onset
• extra glutamine- polyglutamine
• neurological condition - brain problem Motor Cognitive Can’t be created by gene therapy, but gene editing.