Download Section 6 Gene diagnosis

Section 6
Gene diagnosis
• Gene diagnosis is a process which examines the genetic
material contained inside a person’s cells to determine if
the person has or will develop a certain desease.
• Most diseases, if not all, are concerning abnormal
changes of genes.
Characteristics:
• Highly specific
• Highly sensitive
• Highly directed
• Even before the occurrence of illness
Techniques used in gene diagnosis

NA hybridization

PCR

DNA sequencing

Polymorphism analysis
Restriction fragment length polymorphism
The basic technique for detecting RFLPs involves fragmenting a
sample of DNA by a restriction enzyme, which can recognize and cut
DNA wherever a specific short sequence occurs, in a process known
as a restriction digest. The resulting DNA fragments are then
separated by length through agarose gel electrophoresis, and
transferred to a membrane via the Southern blot procedure.
Hybridization of the membrane to a labeled DNA probe then
determines the length of the fragments which are complementary to the
probe. A RFLP occurs when the length of a detected fragment varies
between individuals.
Sickle anemia patient’s genome
MstⅡcutting site (GCTNAGG)
5´
3´
1.15kb
×
Gene of normal hemoglobin beta subunit
mutation (GCTNTGG)
5´
3´
1.35kb
Mutated gene
正常人
Normal
突变携带者
患者
Carrier
Patients
PCR single strand conformation
polymorphism, PCR-SSCP
Single strand conformation polymorphism
(SSCP) is defined as conformational difference of
single stranded nucleotide sequences of identical
length as induced by differences in the sequences
under certain experimental conditions. This
property allows to distinguish the sequences by
native gel electrophoresis, which separates the
different conformations.
PCR-SSCP
Normal PCR product
mutationed PCR product
denaturation
denaturation
Native PAGE
Normal Carrier Patients
Applications of gene diagnosis
• Inherited diseases( Phenylketonuria)
• Diagnosis of cancer
• Diagnosis of infectious diseases
• Forensic medicine
• Abnormal gene expression
Section 7
Gene therapy
• Gene therapy is the way to transfer genetic
material which exerts the biological function into
cells of patients to treat the disease.
• A normal gene, recombinant DNA, RNA,
synthetic oligonucleotides can be used.
1. Therapeutic strategies
 gene correction
 gene replacement
 gene augmentation
 gene inactivation
 Killing of specific cells
The suicide gene therapy strategy
2. Gene therapy may be classified into two types
 Germ line gene therapy
Germ cells, i.e., sperm or eggs, are modified by the introduction of
functional genes, which are ordinarily integrated into their genomes.
Therefore, the change due to therapy would be heritable and would be
passed on to later generations. This new approach, theoretically, should
be highly effective in counteracting genetic disorders and hereditary
diseases. However, many jurisdictions prohibit this for application in
human beings, at least for the present, for a variety of technical and ethical
reasons.
 Somatic gene therapy
Therapeutic genes are transferred into the somatic cells of a patient.
Any modifications and effects will be restricted to the individual patient only,
and will not be inherited by the patient's offspring.
3. Basic process of gene therapy
 Selection of a therapeutic gene
wild type gene
 Selection of vehicles
virus(retrovirus, adenovirus, adeno-associated virus…)
 Selection of target cells
somatic cells
 Gene transfer
directly, indirectly
Gene transfer strategies
Gene therapy introduced by virus indirectly
截至2004 年6 月底，全世界范围内基因治疗的临床试验方案
Severe combined immunodeficiency, SCID
(ADA syndrome)
SCID is a genetic disorder in which both B cells and T cells of the
adaptive immune system are crippled, due to a defect in adenosine
deaminase (ADA) gene, necessary for the breakdown of purines.
Lack of ADA causes accumulation of dATP. This metabolite will inhibit
the activity of ribonucleotide reductase, the enzyme that reduces
ribonucleotides to generate deoxyribonucleotides. The effectiveness of
the immune system depends upon lymphocyte proliferation and hence
dNTP synthesis. Without functional ribonucleotide reductase,
lymphocyte proliferation is inhibited and the immune system is
compromised.
SCID is a severe form of heritable immunodeficiency. It is also
known as the “bubble boy" disease because its victims are extremely
vulnerable to infectious diseases.
In 1990, Ashanthi's gene therapy
procedure, doctors removed T cells from
the child's body, let the cells grow in the
laboratory, inserted the missing ADA gene
into the cells, and then infused the
genetically modified T cells back into the
patient's bloodstream. The T cells treated
genetically only work for a few months,
after which the process must be repeated.
As of early 2007, she was still in good
health, and she was attending college.