Section 6

Gene diagnosis

Characteristics:

• Highly specific

• Highly sensitive

• Highly directed

• Even before the occurrence of illness

• 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 chang

es of genes.

Techniques used in gene diagnosis

NA hybridization

PCR

DNA sequencing

Polymorphism analysis

The basic technique for detecting RFLPs involves fragmenting a sa

mple of DNA by a restriction enzyme, which can recognize and cut DN

A wherever a specific short sequence occurs, in a process known as a

restriction digest. The resulting DNA fragments are then separated by l

ength through agarose gel electrophoresis, and transferred to a membr

ane via the Southern blot procedure. Hybridization of the membrane to

a labeled DNA probe then determines the length of the fragments whic

h are complementary to the probe. A RFLP occurs when the length of

a detected fragment varies between individuals.

Restriction fragment length polymorphism

×

Mst cutting site (GCTNAGG)Ⅱ

5´ 3´

Gene of normal hemoglobin beta subunit

5´ 3´

Mutated gene

1.15kb

1.35kb

Sickle anemia patient’s genome

mutation (GCTNTGG)

正常人 突变携带者 患者Normal Carrier Patients

PCR single strand conformation polymorphism, PCR-SSCP

Single strand conformation polymorphism (S

SCP) is defined as conformational difference of si

ngle stranded nucleotide sequences of identical le

ngth as induced by differences in the sequences

under certain experimental conditions. This prope

rty allows to distinguish the sequences by native

gel electrophoresis, which separates the different

conformations.

Normal PCR product mutationed PCR product

denaturation denaturation

Native PAGE

Normal Carrier

Patients

PCR-SSCP

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 mate

rial which exerts the biological function into cells

of patients to treat the disease.

• A normal gene, recombinant DNA, RNA, syntheti

c oligonucleotides can be used.

1. Therapeutic strategies

gene correction

gene replacement

gene augmentation

gene inactivation

Killing of specific cells

The suicide gene therapy strategy

Germ line gene therapy

Germ cells, i.e., sperm or eggs, are modified by the introduction of funct

ional genes, which are ordinarily integrated into their genomes. Therefore,

the change due to therapy would be heritable and would be passed on to l

ater generations. This new approach, theoretically, should be highly effecti

ve 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. An

y modifications and effects will be restricted to the individual patient only, a

nd will not be inherited by the patient's offspring.

2. Gene therapy may be classified into two types

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 a

daptive immune system are crippled, due to a defect in adenosine dea

minase (ADA) gene, necessary for the breakdown of purines. Lack of

ADA causes accumulation of dATP. This metabolite will inhibit the acti

vity of ribonucleotide reductase, the enzyme that reduces ribonucleotid

es to generate deoxyribonucleotides. The effectiveness of the immune

system depends upon lymphocyte proliferation and hence dNTP synth

esis. Without functional ribonucleotide reductase, lymphocyte prolifera

tion is inhibited and the immune system is compromised.

SCID is a severe form of heritable immunodeficiency. It is also kno

wn as the “bubble boy" disease because its victims are extremely vuln

erable to infectious diseases.

In 1990, Ashanthi's gene therapy proce

dure, doctors removed T cells from the chi

ld's body, let the cells grow in the laborato

ry, inserted the missing ADA gene into the

cells, and then infused the genetically mo

dified T cells back into the patient's bloods

tream. The T cells treated genetically only

work for a few months, after which the pro

cess must be repeated. As of early 2007,

she was still in good health, and she was

attending college.