Advances in Genetic Technology

Class Notes

Make sure you study this along with our first PowerPoint on Transgenics and your class Article notes

When we talk about cloning, are we always talking about the same

thing?

• recombinant DNA cloning (transfer of gene of interest to a vector like bacteria or a virus which will incorporate in all future DNA)

• reproductive cloning (transfer of nuclear material from a somatic cell of one organism to an egg of another that has had its nucleus removed, resulting in an offspring like the donor)

• therapeutic cloning (also known as embryo cloning is designed to create stem cells for use in treating disease)

How is a mammal cloned (reproductive

cloning)?

Is this possible In humans?

There are lots of efficiency problems. It takes hundreds of tries to get one clone, even in a sheep.

What is PCR and why do we need it?

• Polymerase chain reaction (PCR) is a fast and inexpensive technique used to "amplify" - copy - small segments of DNA.

• It is used wherever large amounts of DNA are needed. Most often there is only a small sample of DNA to begin with.

• Examples of its use: DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders.

Steps to PCR mimic what happens in replication

If you run about 35 cycles and end up with billions of copies of the original DNA

What is a Short Tandem Repeat (STR)Polymorphism and how is it used?

• STRs are short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times.

• These are inherited regions of our DNA that can vary from person to person.

• These are located at the same place on the chromosomes but they vary (polymorphism) in length from person to person.

• It is highly unlikely that an individual would have the same number of repeats as another individual at the most common13 STR sites used by criminal justice to distinguish individuals.

These loci on the chromosomes are the location of STRs and are used to distinguish one individual from another. PCR can be used to make many copies of the DNA segments with STRs for forensic analysis.

Electrophoresis is used in separating DNA fragments

• Gel electrophoresis is a method of separating large molecules (such as DNA fragments or proteins) from a mixture of similar molecules.

• An electric current is passed through a gel containing the DNA mixture, and each kind of molecule travels through the medium at a different rate, depending on its electrical charge and size.

• The gel acts as a sieve. DNA moves at different rates because larger molecules move through the gel matrix slower than smaller molecules.

The Human Genome Project(approx. 1990-2003)

Project goals were to: • identify all the approximately 20,000-25,000

genes in human DNA, • determine the sequences of the 3 billion chemical

base pairs that make up human DNA, • store this information in databases, • improve tools for data analysis, • transfer related technologies to the private

sector

Applications of the Data from the Human Genome Project

• Molecular medicine • Energy sources and environmental applications • Risk assessment • Bioarchaeology, anthropology, evolution, and

human migration • DNA forensics (identification) • Agriculture, livestock breeding, and

bioprocessing

What is Gene Therapy?Gene therapy is designed to introduce beneficial versions of a gene into an afflicted individual

for therapeutic purposes.How does it work?• For gene therapy to be permanent, the cells that receive the normal allele must be ones

that multiply throughout the patient's life. • In most gene therapy studies, a "normal" gene is inserted into the genome to replace an

"abnormal," disease-causing gene using a vector. • Currently, the most common vector is a virus that has been genetically altered to carry

normal human DNA.

Two mechanisms: direct into the tissue or through the use of stem cells (unspecialized cells) to carry the gene into the body.

How do small RNAs (microRNA, RNAi) impact the expression of traits?

• These are short nucleotide sequences that bind to the complementary RNA made during transcription and they usually “silence” the gene.

• MicroRNAs target about 60% of all genes. They are abundant in all human cells and are able to repress hundreds of targets each.

When the microRNA interferes with translation the protein will not be produced and therefore the gene is “silenced” or not expressed.

How can we determine if a gene will “express” itself?

A microarray is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.

With microarray technology.

Scientists can determine, in a single experiment, the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array.

Often they are looking for Single Nucleotide Polymorphisms (SNPs)

Compare a normal healthy sample with the patients sample

 GREEN represents Control DNA,  RED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA. YELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA. BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA. 

Different types of microarrays have different applications

In Brief: Microarray Applications

Microarray type Application

CGH Tumor classification, risk assessment, and prognosis prediction

Expression analysis Drug development, drug response, and therapy development

Mutation/Polymorphism analysis Drug development, therapy development, and tracking disease progression

What is a SNP?

• It is a specific type of mutations in DNA. This small variation is enough to cause a disease or disability.

• When researchers use microarrays to detect mutations or polymorphisms in a gene sequence, the target, or immobilized DNA, is usually that of a single gene.

• In this case though, the target sequence placed on any given spot within the array will differ from that of other spots in the same microarray, sometimes by only one or a few specific nucleotides.

• One type of sequence commonly used in this type of analysis is called a Single Nucleotide Polymorphism, or SNP, a small genetic change or variation that can occur within a person's DNA sequence.

Last but not least, what does epigenetics have to do with this?

• Epigenetics influence the expression of traits.

• It is now clear that your destiny is not limited by your genes alone. There are plenty of non-genetic influences on expression of your traits.

• It is through epigenetic marks that environmental factors like diet, stress and prenatal nutrition can make an imprint on genes that are passed from one generation to the next

•Epigenetic mechanisms include methylation and acetylation of nucleotides which cause the DNA to bind tightly or more loosely around the histones.

•The ultimate result is the transcription of information onto mRNA or not.