Gene Diagnosis E-mail: haiyanhuang@shmu.edu.cn

Central Dogma of Biology Disease

Definition of Gene Diagnosis Analysis of DNA and RNA in order to detect heritable disease-related genotypes, mutations, modifications, karyotypes, or micro-organisms for clinical purposes. (DNA Diagnosis) (RNA Diagnosis)

u u u u u u u Blood samples - the most widely used source of DNA from adults. Mouthwashes or buccal scrapes - being noninvasive, they are especially favored for population screening programs. Mouthwashes yield sufficient DNA for a few dozen tests, and by using whole genome amplification more extensive testing of a single sample may be possible. Chorionic villus biopsy samples - the best source of fetal DNA (better than amniocentesis specimens). One or two cells removed from eight-cell stage embryos, for pre-implantation diagnosis after in vitro fertilization. Hair, semen, etc. for criminal investigations. Archived pathological specimens, for typing dead people when no DNA has been stored, or testing tumors for genetic changes. Only short sequences, 250 bp or less, can be reliably amplified from fixed tissue specimens. Guthrie cards - these are the cards on which a spot of dried blood is sent to a laboratory for neonatal screening for phenylketonuria (PKU) in the UK and elsewhere; not all the blood spot is used for the screening test. They are a possible source of DNA from a dead child.

u u u u u

Polymerase Chain Reaction (PCR) Kary Banks Mullis Micheal Smith Kary received a Nobel Prize in chemistry in 1993

PCR

5 A T G C C G A T 3 T A C G G C 5 A T G C G T A 3 U A C G C A U 5 A U G C U A C G 3 U A C G A U G C

Southern blotting

Northern blotting

restriction fragment length polymorphism RFLP

restriction fragment length polymorphism RFLP Schematic for RFLP by cleavage site loss Analysis and inheritance of allelic RFLP fragments Schematic for RFLP by VNTR length variation

Amplification refractory mutation system ARMS

GGGG GGGG GGGG CCCC PCR GGGG CCCC GGGG CCCC GGGG CCCC DNA : >A/T>G/C

Single Strand Conformation Polymorphism SSCP is the electrophoretic separation of single-stranded nucleic acids based on subtle differences in sequence (often a single base pair) which results in a different secondary structure and a measurable difference in mobility through a gel.

Single Strand Conformation Polymorphism +/+ -/+ -/-

Gene Chip

Wild type Gene Chip mutant

Cytogeneticists can now go "FISH-ing" for chromosomal abnormalities, which are deletions and duplications that can cause disease. How exactly does FISH work?

Principles of fluorescence in situ hybridization (FISH)

Principles of fluorescence in situ hybridization (FISH)

( Whole Chromosome Painting)

Application of gene diagnosis

Types of Genetic Diseases Single-gene/monogenic Genetic Diseases. sickle cell anemia, cystic fibrosis, Aicardi Syndrome, Huntington s disease Multifactorial/Polygonic Genetic Diseases. Alzheimer, diabetes, obesity, arthritis,cancer, high blood pressure Chromosomal Genetic Diseases trisomy21 Mitochondrial Genetic Diseases

Maternal Age 15-24 < Risk of chromosomal abnormality 1/500 Risk of Down s Syndrome 1/1500 25-29 1/385 1/1100 35 40 45 1/178 1/63 1/18 1/350 1/100 1/25

sickle cell anemia

(HbS) (HbS) 5 7 probe A 1.15Kb 0.2Kb A A A S S S 1.35 Kb 1.15 Kb S 1.35 Kb 0.2Kb 0.20 Kb

Cystic fibrosis

Cystic fibrosis

Chromosomal Genetic Diseases Down syndrome

LSI 13 Spectrum Green probe LSI 21 Spectrum Orange probe G-banded karyotype of a trisomy 21 female Fluorescent in situ hybridization (FISH)

Pm 3 43 50 13 6 47 60 52 1 2 3 4 5 6 7 8 bp 535 113

Doctor Robert Edwards

In vitro fertilization (IVF)

preimplantation u Preimplanation Genetic Diagnosis (PGD), first developed in the 1980's, is the application of genetic testing on a live embryo to determine the presence, absence or change in a specific gene or chromosome prior to the placement of the embryo in the womb.

Preimplantation genetic diagnosis involves screening embryos for genetic defects u The first step to PGD is the creation of embryos outside the body by in vitro fertilisation (IVF). These embryos are cultured until they are between 6-10 cells in size so that one or two cells can be removed (cell biopsy) for genetic analysis. The removal of these cells does not appear to be detrimental to the development of the embryo

Principles of preimplantation genetic diagnosis Currently, PGD can be used to detect many disorders, including cystic fibrosis, Down syndrome, Tay-Sachs disease and hemophilia A

Target Sequence DNA Template DNA Template First set primer Target Sequence Target Sequence DNA Template First PCR Run Majority of PCR Product Target Sequence Target Sequence Second set primer Target Sequence Second PCR Run PCR Product