Cell Cloning Part 3 - 5

Steps of cloning (YFG) using pBR322

Must use PstI to cut your gene and plasmid

Cut plasmid and DNA using PstI

Use DNA ligase to from the recombinant plasmid, Am gene non-functional

Transform the plasmid into the E. coli cells

Spread the cells onto a plate with tet. and incubate overnight to eliminate the cells with no plasmids

Using replica plating, spread colonies onto separate agar plates containing Ampicillin

Any of the colonies, which do not grow on the agar containing tetracycline, can be recovered from the ampicillin plate (master plate)

Alkaline Lysis

Alkaline Lysis

The most straightforward and reliable techniques for purification of plasmid DNA

A quick method

This procedure is often described as a “miniprep”

It yields about 1 μg of DNA, which is enough for many analytical purposes

What produces protein

Give the bacteria a chemical signal that instructs them to make the target protein

The bacteria serve as miniature “factories”

• e.g. Human insulin

• Expressing human gene in bacteria

Shine Dalgarno

Plays a role in initiation of translation (ribosomal binding site)

Is it important that the enhancer and TATA sequence might be left behind due to the placement of the Shine Dalgarno?

No

The plasmid provides the needed reeded regulatory signals

Restriction Mapping

The process of obtaining structural information on a piece of DNA using restriction enzymes

Used to map an unknown segment of DNA

Used to analyze recombinant closes

• after purifying the plasmid DNA from individual clones

• Cut plasmid with restriction enzymes, and analyze the sizes of the fragments produced

Plasmid Structural info

Size of plasmid

Restriction sites

Steps of restriction mapping

Cut the plasmid DNA with various restriction enzymes

Run Gel Electrophoresis

Analyze the sizes of the fragments produced to obtain structural information

Gel Electrophoresis

To separate the desired DNA based on size after using the restriction enzymes

DNA: Negatively charged (phosphate backbone)

1. DNA molecules are separated according to their size in a gel

2. Actual size is estimated by comparison with marker DNAs of known size

What would be the sizes of the fragments that you would expect to find if you

cut this plasmid with (a) BamHI, (b)EcoRI, (c) both BamHI and EcoRI together?

(a) If you cut with BamHI you would expect two fragments of 0.5 kb and 2.8 kb.

(b) If you cut with EcoRI you would expect a single fragment of 3.3 kb.

(c) If you use both restriction enzymes together you would expect three fragments of 0.5, 0.8 and 2 kb.

Create a plasmid map using the following electrophoresis data

See that there are 3 EcoRI (1st column)

• 1 10kbp

• 1 30kbp

• 1 60 kbp

See that there is 1 HindII (2nd conlum)

• Split the 60kpd section into: (3rd column)

  • 20kpd

  • 40kpb

Create a plasmid map using the following electrophoresis data

1st column tells us

• 1 EcoRI

2nd column tells us

• 3 BemHI

  • 1 11kb

  • 1 6 kb

  • 1 3 kbp

3rd column tells us

• The EcoRI splits the 11kd section

  • 3 kb

  • 8 kb

Applications of Recombinant DNA Technology

Medical Applications

Agricultural Applications

Industrial Applications

Medical Applications of Recombinant DNA Technology

Gene therapy

Production of transgenic organisms

Diagnosis of genetic disease

Gene Therapy

Primarily an experimental technique that uses genes to treat or prevent disease (NIH)

Is Gene Therapies FDA approved

The US FDA has approved a total of 34 cellular and gene therapy products within the US

LUXTRNA (2017), Spark Therapeutics, Inc

First FDA approved gene therapy product

Recombinant AAV2 delivers RPE65 to treat patients with RPE65-mutation-associated retinal dystrophy (inherited).

LYFGENIA (2023), Bluebird Bio, Inc

Cell (autologous, modified): Hematopoietic stem cells engineered with a lentivirus, BB305, expressing modified ßA-globin gene.

Treats patients with certain kinds of sickle cell disease

CASGEVY (2023), Vertex Pharmaceuticals, Inc

Cell (autologous, modified): CD34+ hematopoietic stem cells engineered with electroporation of CRISPR/Cas9 RNP complexes to decrease expression of BCL11A to increase fetal hemoglobin production.

Treats patients with certain kinds of sickle cell disease.

Approaches to Gene Therapy

1. Inactivate a mutated gene that is functioning improperly (Gene inhibition therapy)

2. Replace a mutated gene that causes disease with a healthy copy of the gene

3. Introduce a new gene into the body to help fight a disease

Gene inhibition Therapy

Inactivate a mutated gene that is functioning improperly

Gene replacement therapy

Replace a mutated gene that causes disease with a healthy copy of the gene

Gene introduction therapy

Introduce a new gene into the body to help fight a disease

Gene Therapy Procedure

A vector is genetically engineered to deliver the gene (viruses)

The viruses are modified so they can't cause disease when used in people

Retroviruses

Integrate their genetic material (including the new gene) into a chromosome in the human cell

Adenoviruses

Introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome

Adenovirsus Procedure

1. Vector can be injected or given intravenously (IV) directly into a specific tissue in the body, where it is taken up by individual cells

2. A sample of the patient's cells can be removed and exposed to the vector in a laboratory setting

   • The cells containing the vector are then returned to the patient

Applications of Gene Therapy

A promising treatment option for a number of diseases

• Cancer, cystic fibrosis, heart disease, and diabetes

Limitations of Gene Therapy

Remains an area of active research and study

• long-term effects under investigation

• there are risks

Risks of Gene Therapy

Unintended Immune Responses

Off-Target Effects

• Targeting the wrong cells

• Unintended locations in the genome

Infection caused by the virus.

Risk of Tumorigenesis

Ethical & Social Concerns

Unintended Immune Responses

The body may see the newly introduced therapeutic gene or the vector used to

deliver it as intruders and attack the. This may cause inflammation and, in severe

cases, organ failure.

Off-Target Effects - Targeting the Wrong Cells

Because viruses can affect more than one type of cells, it's possible that the altered viruses may infect additional cells — not just the targeted cells containing mutated genes. If this happens, healthy cells may be damaged, causing other illness or diseases, such as cancer.

Off-Target Effects - Unintended locations

The possibility of the therapeutic gene integrating into unintended locations in the genome, potentially disrupting normal gene function

Infection caused by the virus

Viral Replication: It's possible that once introduced into the body, the viruses may recover their original ability to replicate to cause disease

Risk of Tumorigenesis

Possibility of causing a tumor: Insertional mutagenesis, where the integration of the therapeutic gene into the genome increases the risk of tumor formation.

Types of Gene Therapy

Somatic Therapy

Germ-line Therapy

Somatic Therapy

Healthy copy of a gene is inserted into somatic cells

• Affects only the targeted cells in the patient

• not passed on to future generations

Germ-line Therapy

Treats cells that make eggs and sperm

• Permanent changes that are passed down to subsequent generations

• The goal would be to change the eventual child's genetic inheritance

Ethical Issues

First Human Gene Therapy Experiment

Carried out at NIH in 1990

The trial aimed to treat a genetic disorder called severe combined immunodeficiency (SCID)

Specifically the form caused by a deficiency of the adenosine deaminase (ADA) enzyme. The condition is commonly known as ADA-SCID.

the initial results showed some improvement in immune function

the overall success was limited, and the treatment did not provide a complete cure.

this groundbreaking experiment marked the beginning of human gene therapy research.

Production of Transgenic Animals

Recombinant DNA technology has made it possible to create transgenic animals to produce human proteins

Transgenic Animals

Animals that are genetically modified

Important tools for researching human disease

Mice have been genetically modified to naturally produce human antibodies for use as therapeutics

Diagnosis of Genetic Disease

Can provide precise diagnostic information about genetic diseases, allowing appropriate counselling, and indicating future directions for research on therapeutic intervention, e.g. gene therapy.

• Genetic screening

• Genetic sequencing

Agricultural Applications of Recombinant DNA Technology

Improving yields

Resistance to disease

Way of using recombinant DNA technology to improve yields

A faster rate of growth

An increase in the quantity of the food produced by plants

Nitrogen Fixing Gene

N2 is vital nutrients for most plants

Plants are unable to utilize N2, which is abundant in the atmosphere

Fixation

Synthetic nitrogen fertilizer, Problems

Solution

N2 Fixation

Convert N2 to a useable form like ammonia

Synthetic Nitrogen Fertilizer

Biologically reactive nitrogen is therefore routinely supplied to crops as synthetic nitrogen fertilizer

Synthetic Nitrogen Fertilizer Problems and Solution

The extensive use of synthetic fertilizer in agriculture has substantial environmental costs

Synthetic fertilizers lead to pollution

Many of these problems could be avoided if plants could be engineered to fix nitrogen directly from air

Procedure to make plants fix nitrogen from the air

1. Nitrogen fixing genes can be cloned in E. coli and placed into cells in crop

   This would enable these crops to fertilize themselves effectively, saving much money and increasing yields

How to make crops resistance to disease

Genes are introduced into crops, causing them to produce their own pesticides

Example of making crops resistance to disease

Place a gene into tomato plant that produces a protein, which kills tomato worms

When modified tomato plants and normal plants were grown together in a laboratory and deliberately infected with worms

The normal plants were stripped of their leaves, but the modified plants were unaffected

Industrial Application of Recombinant DNA Technology

Bioprocessing

Production of Drugs and Food

DNA Sequencing

The Polymerase Chair Reaction (PCR)

PRC

Polymerase Chair Reaction

An in vitro technique for synthesizing many copies of DNA

Generates thousands to millions of copies of a particular DNA sequences

PCR-Required Materials

• DNA polymerase

• A single-stranded DNA template

• PCR Primers

• Deoxynucleotide triphosphates (dNTPs)

DNA Template (for PCR)

A nucleotide sequence that directs the synthesis of a sequence complementary to it

Taq DNA polymerase

Enzyme that copies the DNA

PCR Primers

A strand of DNA bases that enables DNA to be replicated

dNTPs

Single units of the bases A, T, G, and C,

Building blocks for new DNA strands

First step of PCR

PCR primers design is the first step

Synthesis cannot begin unless there is already a short double- stranded region to be copied

If the matching occurs, then DNA polymerase can bind the dNTPs

PCR Steps

Denaturation

Annealing Extension

Outcome of PCR

Exponential increase in the number of target molecules in successive cycles

After 22 cycles you would expect to have over a million target molecules

2^22 = 4,194,304

How many cycles of PCR are done in practice

25-35 cycles are usually preformed to ensure sufficient amplification of the desired region of the template

Advantages of PCR

PCR is a straightforward technique

Amplify DNA starting with a very small sample (as little as one molecule), you can make large quantities of DNA

It allows the amplification of a specific region of DNA without reliance on pre- existing restriction sites

Limits of PCR

DNA sequence information is needed before you begin

The length of the region that can be amplified:

• PCR works well over short stretches of DNA up to about 2 kb

• New systems make it possible to amplify target regions of up to 20 kb

DNA Sequencing

Determine the order of the nucleotides that make up a DNA

Sanger Sequencing

Most commonly used technique for sequencing DNA

Depends on PCR

Sanger sequencing gives high-quality sequence for stretches of DNA (up to about 900 base pairs)

It's typically used to sequence individual pieces of DNA, such as bacterial plasmids

Sanger sequencing is expensive and inefficient for larger-scale projects, such as the sequencing of an entire genome

Sanger Sequencing Materials

A single-stranded DNA Template

A DNA primer

A DNA polymerase

Deoxynucleoside triphosphates (dNTPs)

Modified di-deoxynucleotide triphosphates (ddNTPs)

Modified di-deoxynucleotide triphosphates (ddNTPs) (Sanger Sequencing)

The ddNTPs are fluorescently labeled for detection

Each base is labeled differently

Lack the 3'-OH group required for the formation of a phosphodiester bond between two nucleotides

Sanger Sequencing: Steps

Many DNA polymerases attempting to copy many copies of the DNA template strand

Eventually we will have a dideoxy-terminated strand corresponding to every possible nucleotide in the original DNA strand

next-generation sequencing (NGS)

To sequence an entire genome efficiently and cost-effectively, high-throughput sequencing technologies, also known as next-generation sequencing (NGS), are commonly used.

These technologies allow for the simultaneous sequencing of millions of DNA fragments, enabling rapid and comprehensive genome analysis.