INSERTING A GENE INTO A PLASMID VECTOR

                          WHAT IS A VECTOR?

A vector is a small circular piece of DNA used to artificially carry genetic material into another cell.

For example; plasmids ,viruses ,liposomes

Plasmids are most commonly used as vectors.

Plasmids are normally found in bacterial cells and often contain genes for antibiotic resistance and can be exchanged between bacteria of the same species or of different species through bacterial conjugation.

Bacterial Conjugation is the transfer of genetic material through direct cell to cell contact or by a bridge-like connection between two cells

Why are plasmids used in gene cloning?

Plasmids have properties that allow them to be used for this process. They are listed below:

An Origin Of Replication- This allows foreign (desirable) DNA to be replicated within the host cells.

Marker Genes- This allows identification of cells that have taken up the plasmid

Several single target sites for different restriction enzymes

Low Molecular Mass- This is so that it is taken up easily by bacteria

                           How are Plasmids used as vectors?

A piece of DNA is inserted into the plasmid and the plasmid is used to take the DNA into the bacterial cell.

The bacteria containing the plasmids are treated with enzymes to break down their cell walls

The naked bacteria is then spun at high speed in a centrifuge to separate the larger bacterial chromosomes from the much smaller plasmids

The circular DNA of the plasmid is cut open using a restriction enzyme and the same enzyme is used to cut the gene that should be used so that the sticky ends will be complementary.

 The sticky ends of both the gene and the plasmid DNA are linked together using Ligase Enzyme.

The end product is called Recombinant DNA

Recombinant DNA is DNA made by joining pieces of DNA from two or more sources/species

Getting Plasmids Into Bacteria

The bacteria is first treated by putting them into a highly concentrated solution of Calcium ions.

It is then cooled and given a heat shock to increase the chances of plasmids passing through the cell surface membrane

Only a small proportion of the bacteria actually take up the plasmids and when this occurs it is called Transformation.

Identifying bacteria with recombinant DNA

To identify which bacteria have been transformed successfully, they are spread on agar plates containing an antibiotic.

The bacteria with the recombinant DNA would be able to grow on the agar containing the antibiotic. This is because the plasmid with the recombinant DNA may contain genes for resistance to that antibiotic.

The DNA polymerase in the bacteria with the recombinant DNA copies the plasmids and divides by binary fission so that each daughter cell has copies of the gene. The bacteria then transcribe the new gene and translates it to give the required gene product. For example; insulin

Synthesizing Genes For Insulin

Sometimes locating and isolating the gene for human insulin can be difficult.

So instead of cutting out the gene, genetic engineers extract the mRNA which codes for insulin from the Beta cells in the pancreas and incubate it with the enzyme Reverse Transcriptase.

This enzyme reverses transcription by using mRNA as a template to make single-stranded DNA molecules which are later converted to double-stranded DNA molecules by using DNA polymerase to assemble nucleotides to form the complementary strand.

This is then inserted into a plasmid to transform a bacterium.

GENETIC SCREENING

GENETIC SCREENING and ITS ETHICS

What is Genetic Screening?

Genetic screening refers to testing an embryo, fetus or adult to find out if a particular allele is present. The body can be screened for things like cancers, Duchenne muscular dystrophy, thalassaemia, haemophilia, sickle cell and Huntington’s disease.                                                                                                                                                                                    

Process of Genetic Screening                                      
1. Genetic tests are performed on a sample of blood, hair, skin, amniotic fluid or other tissue                                                     2. For example, a procedure called a buccal smear uses a small brush or cotton swab to collect a sample of cells from the inside surface of the cheek                                                    
c.The sample is sent to a laboratory where technicians look for specific changes in chromosomes, DNA, or proteins, depending on the suspected disorder.                                               
d.The laboratory reports the test results in writing to a person's doctor or genetic counsellor, or directly to the patient if requested.  
                                                                                                                                                                                   
How is Genetic screening used to screen for sickle cell anaemia                                                                                              1.A blood test can check for haemoglobin S — the defective form of haemoglobin that underlies sickle cell anaemia.             2.In adults, a blood sample is drawn from a vein in the arm. In young children and babies, the blood sample is usually collected from a finger or heel.                                          
3.The sample is then sent to a laboratory, where it's screened for haemoglobin- S.                                                             
4.If the screening test is negative, there is no sickle cell gene present. If the screening test is positive, further tests will be done to determine whether one or two sickle cell genes are present.

                    Advantages of genetic screening                                 
Test results can provide a sense of relief from uncertainty and help people make informed decisions about managing their health care. New born screening can identify genetic disorders early in life so treatment can be started as early as possible.

WHAT IS A DESIGNER BABY?
A designer baby is a baby with artificially changed genetic information. It is officially known as pre-implantation genetic diagnosis (PGD).Some traits that can be altered for designer babies are;

a.The gender of the baby

b.The outward looks of the baby

c.Reduce the baby’s chances of falling sick or getting affected with a particular disease

d.Change the baby’s overall personality

e.Alter and improve the baby’s overall levels of intelligence

                     HOW ARE DESIGNER BABIES MADE?

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) mechanism

CRISPR “spacer” sequences are transcribed into short RNA sequences capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the CRISPR system – binds to the DNA and cuts it, shutting the targeted gene off. Using modified versions of Cas9, researchers can activate gene expression instead of cutting the DNA.

  Pros:                                                                                                                                                                                    1.Increases life expectancy for up to 30 years                                                                                                                                      2.Reduces chances of various genetic diseases                                                                                                                                        3.You call the shots                                                                                                                                                               4.Eliminates chances of diseases in further generations

Cons:                                                                                                                                                                                                              1.Moral and ethical issues                                                                                                                                                2.Violation of your baby’s rights                                                                                                                                                           3.Not an error-free process                                                                                                                                                           4.May accidentally give rise to new forms of illnesses that scientists are not yet aware of                                                            5.Is not affordable by all hence will create a class divide where only the rich can afford designer babies                                                                                                                            

ETHICAL ISSUES                                                                                 
1.Creating a race of designer or non-designer babies could lead to a dystopian world, in which the designer babies, who are created by modifying the genes, will turn into a superior race and look down upon others who were born without any genetic modification.                                                                                                                                                                                2.Gender-specific abortions                                                                                                                                                                3.Many think that sex pre-selection is unethical                                                                                                                            4. Amniocentesis and chronic villus sampling                                                                                                                             5.Reproduction is being turned into a ‘baby market’                                                                                                                                             6.If designer babies become fashionable, this trend will be difficult to control                                                                 

GENETIC MARKERS AND PROMOTERS

                         GENETIC       MARKER                                                                                     WHAT IS A GENETIC MARKER?A genetic marker is a gene sequence with a known location on a chromosome that can be used to identify species. It can be used to study the genetics of an organism.

TYPES OF GENETIC MARKERS                                                                                                             1.Restriction Fragment length polymorphism(RFLP).                                                                              2.Simple sequence length polymorphism(SSLP).                                                                                     3.Amplified fragment Length Polymorphism(AFLP).                                                                              4.Variable number tandem repeat(VNTR)

What is polymorphism? It is a term that describes multiple forms of a single gene that exists in an individual

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)                                             This was the first DNA based marker developed.                                                                                      It is a method that allows individuals to be identified, based on the pattern of the restriction enzyme, cutting in a specific region of DNA.                                                                                                           They are mainly co-dominant.                                                                                                                   It is used to detect a mutation in a gene

Method of DNA analysis by RFLP

First, a particular region of the DNA is cut with a restriction enzyme (enzyme that cuts the restriction site) Then , separate the DNA fragments using gel electrophoresis

Then it is transferred to a membrane by the southern blot (it is used to detect a specific DNA sequence)

Lastly determine the number of fragments according to their lengths and pattern of fragment sizes.

APPLICATION OF RFLP                                                                                                              1.DNA fingerprinting: helps identify suspects based on the samples taken from crime scenes.               2.Paternity : It is used to determine the father of a child.                                                                           Negative Aspect of RFLP                                                      1.Tedious and slow                                                                                                                                    2.Large amount of DNA Sample is required.                                                                                                                                                                                Positive Aspect of RFLP                              1.Can be easily transferred between labs.

What is VNTR?                                                                                                                                   VNTR is a location in a genome where a short nucleotide sequence is organized as a tandem .It is a part the DNA that is different for every individual. It is found on many chromosomes and the size is about 7 – 10 base pair long

APPLICATION OF VNTR                                                                                                                         1.DNA fingerprinting                                                                                                                                 2. Genetic diversity (total number of genetic characteristics in the makeup of a specie

. Examples of Genetic markers                                                                                                                    a.Green Fluorescent Markers                                                                                                    b.Enzyme Beta glucuronidase

                                                    Green Fluorescent Marker                                                                    a.GFP is found in a jellyfish that lives in cold regions.                                                                            b.GFP monitors gene expression and cell division.

APPLICATION                                                                                                                                1.Jellyfish contains a bio luminescent protein that emits blue light. The GFP converts the blue light to green light. And this is what we see when a jellyfish lights up.                                                              2.GFP does this by absorbing UV Light from the sun and emits as green light.

                          Green Fluorescent Protein                                                                                              a.Comes from bio luminescent jellyfish- Aequorea Victoria.                                                                    b. GFP is widely used as a marker to determine if transformation is effective

                        What is the use of GFP?                                                                                                   1.It allows us to see the inner workings of the cells.                                                                                  2.Easy to locate because it will glow bright green.                                                                  ENZYME Beta    glucuronidase.                                                                                                       1.The enzyme Beta glucuronidase (GUS), originates from E.coli.                                                           2.Any cell incubated with some specific colorless substrate, is transformed into a colored product.   3.It is mainly used to determine the activity of inserted genes in plants.                                  

Why fluorescent Markers are used in place of Antibiotic Resistance.                                     1.They are easier to identify.                                                                                                                     2.It is time saving.                                                                                                                                      3.The resistance gene can be passed to other bacteria.                                                                             4.The antibiotics are no longer effective and require new treatment because of the spread of the antibiotic gene resistance.

                                            PROMOTER                                                                                                                                A promoter is a region of the DNA to which RNA polymerase binds as it starts transcription. For example E.coli make the enzyme Beta galactosidase when growing in a medium with lactose. It maps ends of mRNA onto a DNA. It allows RNA polymerase to bind to DNA, and it ensures that it recognizes which of the two DNA strands is the template strand .It controls the gene expression.Most promoters are located at the 5’ end of the gene. Can be about 100-1000 base pair long. Promoters have specific sequences and determine whether a gene gets transcribed .Without a promoter DNA will not be transcribed.

                              Why a promoter must be transferred with a desired gene

 The promoter acts as a binding site for RNA polymerase, and initiates transcription. This makes it easier for the gene to be inserted into the promoter.                                                                                                                FUNCTION OF PROMOTER

1.RNA polymerase binding site

2.Initiates transcription

3.Control the gene expression

HAEMOGLOBIN

                                         HAEMOGLOBIN                                                         –                                                                                                                          Body cells need a constant supply of oxygen.Oxygen  is transported around the body inside red blood cells.Each haemoglobin molecule is made up of four polypeptides.                                                    Each haem group combine with one oxygen molecule                                                                       –      Hb +4O2  =   HbO8                                                                                                                 Haemoglobin molecules that transport oxygen :                                                                           1.Pick up oxygen at the lungs                                                                                                  2.Release oxygen within respiring tissues                                                                         3.Investigating how Haemoglobin behaves.
       Investing how haemoglobin behaves:

•  ž  Extract samples from blood
•  ž  Expose samples to different partial pressures
• ž      Measure amount of oxygen which combines with each sample of haemoglobin   NOTE:Maximum amount of oxygen that haemoglobin sample can combine with is 100% and therefore said to be saturated .Saturation of haemoglobin with oxygen is therefore measured in percentage and can be plotted against partial pressure of oxygen to obtain a curve.                                                                      

ž  Haemoglobin from lungs carries a lot of oxygen as it reaches a muscle ,it releases around ¾ of oxygen which diffuses out of red blood cell and into muscle where it can be used in respiration.

ž  S –shaped curve.

ž  Shape of dissociation curve can be explained by behaviour of haemoglobin molecule                                                                                                                                                                                 

ž  When an oxygen molecule combine with one haem group , the whole haemoglobin is slightly distorted.                                                                                                                                                                                                

  Bohr   Shift:

      Behaviour of haemoglobin in picking up oxygen at the lungs and readily releasing it when in low  in oxygen partial pressure                                                                                                

ž  Amount of oxygen,the haemoglobin carries is also affected by the partial pressure of carbon dioxide.

ž  It diffuses from cells and into blood plasma and some diffuses into red blood cells.

ž  In cytoplasm of RBCs there is an enzyme carbonic anhydrase that catalyze the reaction:

ž  CO2 + H2O =H2CO3 (carbonic acid )                  [  DISSOLUTION ]

ž  Hydro carbonic acid dissociates=H2CO3 oxygen ion  + Hydrogen carbonate ion             [DISSOCIATION]

ž  Haemoglobin combines with hydrogen ions forming haemoglobinic acid ,HHb. Result of this reaction is in two – fold:

• ž. Haemoglobin ‘mops up’ the hydrogen ions which are formed when CO2 dissolves and dissociates.
• ž       Presence of high partial pressure of carbon dioxide causes haemoglobin to release oxygen.

               Carbon Dioxide Transport

ž  Bohr effect explains one way in which CO2 is carried in the blood .Another product of dissociation of dissolved carbon dioxide is hydrogen carbonate ions.

ž  Hydrogen carbonate ions is formed in cytoplasm of RBCs because of presence of enzyme carbonic anhydrase .

ž  Some CO2 molecules does not dissociate but remain as CO2 molecules and dissovle in blood plasma.

ž  Other CO2 molecules diffuse into RBCs and combine directly with terminal amine groups (-NH2) of some haemoglobin molecules.

                                                                                                

WHAT IS GENE THERAPY?

Substitution of defective genes in a cell with genetically altered genes. Or

Introduction of genetic material to compensate for abnormal genes or to make a beneficial protein.

·         TYPES OF GENE THERAPY                                                                                              GERM or REPRODUCTIVE

Insertion of allele into gametes or early embryo. Allele is passed on to off springs or germ line.

§                                                                                     SOMATIC                                                                                                      Cells are targeted for gene replacement. Correct allele is placed in body cells .Process is corrective for only patient.

§                                                                                             VECTORS:                                                                                    Systems that enable genes to be delivered to cell. They Transfer, Protection and Transcription of gene. Vector should be:

1.       Less expensive

2.       Easy to produce

3.       Purified in large amounts                                                                                                                                                                                     

v  VIRUSES

Genetically engineered so as to not replicate and thrive in huge numbers to kill cells. VIRUSES

Examples are Retroviruses, Lentiviruses, Adeno-associated viruses.

To deliver a gene in a location, use a virus that attacks that location.

v  LIPOSOMES                                                                                                                                                 Small spheres of phospholipid. Fuse up with target cell membrane and release gene into target cell .Trial for introducing normal allele of CFTR gene into liposomes, then sprayed into noses of patients.                                                                                                                                                                        

v  NAKED DNA                                                                                                                                                      Trails for skin, muscular and heart disorders .This method removes the problems associated with using other vectors

REASONS FOR CHOOSING VECTORS

1.       Efficiency; Monogenetic disorders [caused by a single gene], viral vectors are used.

2.       Span of immune response; In short term expressions, liposomes are used.

3.       Depending on size of target cell; if target cells are big, herpes viruses are used.

4.       Depending whether target cells are dividing or non-dividing.                                        CHALLENGES IN CHOOSING VECTORS                                                                                                                                                                                        
      RETROVIRUSES                                                                                                                                                      Patients may develop leukaemia after gene therapy for X-linked Severe Combined Immunodeficiency (SCID).  They randomly insert genes into host’s genome, hence  they insert their genes within another gene or into regulatory sequence of a gene which may activate neighbouring genes ,leading to cancer.                                                                                                                                                                                      LENTIVIRUSES

Inserts gene randomly into a host genome but can be modified to inactivate replication.                         HIV has been disabled to act as such a vector.

·          ADENO-ASSOCIATED VIRUSES

Virus does not insert its gene into the host genome .This is a problem when host cells are short-lived.

·         LIPOSOMES

Received by a few cells lining the nose .Effects only last for a week.                                           

·          NAKED DNA

Injection of naked DNA can be done by gene gun process. This involves a gold particle covered with corrective DNA and inserted into the cell. This process damages cell membrane.

               SOCIAL AND ECONOMIC CONSIDERATIONS OF GENE THERAPY

1.       Due to technology, treatment is very expensive.

2.       Manipulating the genetic make-up of man is unacceptable because altering the gene is similar to interfering with nature.

3.       Unappreciated due to other simpler method; Drugs.eg:PTC124 allows CFTR to be made to relief symptoms of Cystic fibrosis.