genetic disorders and topics molecular diagnosis disorders and molecular diagnosis...
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Genetic disorders and Molecular diagnosis
นายแพทย ดร. ณตพล ศุภณัฐเศรษฐกุล
ภาควิชาพยาธิวิทยา คณะแพทยศาสตร
มหาวิทยาลัยนเรศวร
Email : [email protected]
http://www.uga.edu/srel/kidsdoscience/images/genetics-puppets.gif http://www.uwo.ca/biology/images/undergraduate/chromosome.jpg 27 พฤศจิกายน พ.ศ. 2556
Topics
Introduction Genetic disorders
Single gene disorders Chromosomal disorders
Multifactorial diseases Somatic cell genetic disorders
Molecular diagnosis
http://history.nih.gov/exhibits/nirenberg/images/photos/01_mendel_pu.jpg
http://www.scq.ubc.ca/wp-content/mendel.jpg
Gregor Mendel ค.ศ.1822-1884
http://tesla.desy.de/~rasmus/media/Coherence/PDFs%20and%20high%20resolution/watson%20and%20crick.jpg
James D. Watson 1928 -
Francis H.C. Crick 1916 - 2004
Structure of DNA 1953
Nobel prize in 1962
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http://i61.photobucket.com/albums/h53/mocost/watsoncrick.jpg http://www.sanger.ac.uk/Info/Press/gfx/030414_hgp_300.jpg
1990 –
2003
Structure of DNA
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/16-05-doublehelix.jpg
Nucleotide = Nucleoside + Phosphate Nucleoside = Ribose sugar + Nitrogenous Base
http://th.wikipedia.org/wiki/%E0%B9%84%E0%B8%9F%E0%B8%A5%E0%B9%8C:Nucleotides_1.svg
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“Central Dogma”
DNA RNA Protein
Translation Transcription
Replication
Human Genome (chromosomes)
In Nucleus 3.1 x 109 bp
In Mitochondria 16,569 bp
Gene
Coding Sequences
(Exon)
Non coding Sequences
(Intron)
Non-gene (Extragenetic)
Repetitive DNA
Tandem repeats (satellite, minisatellite, microsatellite)
Interspersed repeats (SINES, LINES)
Non repetitive DNA
10% 90%
<10% >90% 50% 50%
Cell cycle
http://www.cbp.pitt.edu/faculty/yong_wan/images/main_cell_cycle.jpg
http://ghs.gresham.k12.or.us/science/ps/sci/soph/cells/cycle/cycle.gif
http://tainano.com/Molecular%20Biology%20Glossary.files/image040.gif http://homepage.mac.com/enognog/cell%20cycle.jpg
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Mutation หมายถึงกระบวนการที่กอใหเกิดการเปลี่ยนแปลง
ทุกรูปแบบ ที่เกิดขึ้นกับโมเลกุลของ DNA
“Wild type” and “mutant allele” Normal variation, polymorphism, inherited disease Types of mutation
Base substitution => silent mutation, missense, nonsense Insertion => frameshift mutation Deletion => frameshift mutation Chromosomal abnormality
http://www.layevangelism.com/bastxbk/images/mutation.jpg
http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=112100&screenWidth=1280&screenHeight=800
Genetic disorders
Single gene disorders Mendelian inheritance
Autosomal dominant (AD) Autosomal recessive (AR) X-linked recessive (X-linked)
Non-Mendelian inheritance Chromosomal disorders
Numerical chromosomal abnormality Structural chromosomal abnormality
Polygenic or multifactorial diseases or complex genetic disorders Somatic cell genetic disorders (neoplasia)
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Terminology Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance
http://bio1151.nicerweb.com/Locked/media/ch14/14_06PhenotypeVsGenotype_L.jpg
http://naturalsciences.sdsu.edu/ta/classes/lab2.4/trait.jpg
Terminology Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance
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Terminology Locus = position of DNA sequences, or gene on a chromosome Allele = possible alternative form of a given gene DNA polymorphism = the occurrence in a population of two or more alternative genotypes Gene pool = the study of frequency of total alleles on a given locus Population genetics = the study of the genetic composition of populations
http://ghr.nlm.nih.gov/handbook/illustrations/chromosomallocation.jpg Cystic Fibrosis Transmembrane Regulator, CTFR
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Chromo7.gif
Gene loci http://www.geneticsandhealth.com/wp-content/allele.jpg
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Terminology Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance
http://www.genome.gov/Pages/Hyperion/DIR/VIP/Glossary/Illustration/Images/autosome.gif
Terminology Genotype = genetic constitution of an individual Phenotype = observed characteristic Homozygote = identical alleles at a given locus Heterozygote = two different alleles at a given locus Autosomes = any chromosomes other than sex chromosomes Autosomal inheritance = involve any chromosomes other than sex chromosomes Pedigree charts = to illustrate the inheritance
http://www.geneticsandhealth.com/wp-content/pedigree%20image_1.gif
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http://www.geneticsandhealth.com/wp-content/pedigree%20image_2.gif http://www.migeneticsconnection.org/genomics/Family%20History/pedigreeSymbols.jpg
Introduction
Single gene disorders are caused by individual mutant genes There are approximately 11,000 single gene disorders, affecting 1% of the populations Follow Mendelian patterns of inheritance
Autosomal dominant inheritance (AD) Autosomal recessive inheritance (AR) X-linked recessive inheritance (X-linked) X-linked dominant inheritance Y-linked (Holandic) inheritance
A “dominant phenotype” is one that is expressed in heterozygotes, whereas a “recessive trait” is expressed only in homozygotes If the expression of each allele can be detected in the presence of the other, the two alleles are termed “co-dominant”
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http://www.uic.edu/classes/bms/bms655/gfx/pedigree1.gif
Autosomal Dominant
Phenotypically expressed in homozygotes and heterozygotes for that gene There is vertical inheritance (affected child usually has an affected parent) Unaffected family members usually have unaffected partners, and they produce normal children Affected family members, usually have unaffected partners, and they produce a 1:1 ratio of normal and affected children Usually both sexes are equally affected, and they are equally likely to pass on the disease
http://www.sciencemuseum.org.uk/on-line/genes/images/1-3-5-1-4-2-1-1-1-3-0.jpg
http://www.moondragon.org/obgyn/graphics/achondroplasiacomparison.jpg
http://www.commonwealthadoption.org/images/johnston.jpg
Achondroplasia Achondroplasia
Sporadic type and Inherited type (AD) Fibroblast growth factor receptor 3 (FGFR3) gene mutation Abnormality in cartilage formation Affected people have one normal copy of the FGFR3 gene and one mutant copy Two copies of the mutant gene (homozygous) are fatal
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http://www.nhlbi.nih.gov/health/dci/images/Marfan_lowres.gif
http://www.homoeopathiker.de/homoeopathik/syphilis/bilder/marfan3.jpg
Marfan syndrome
Marfan syndrome Antoine Marfan, a french pediatrician, 1896 Fibrillin-1 (FBN1) gene mutation on chromosome 15 Fibrillin-1 is an extracellular matrix glycoprotein, secreted by fibroblasts Structural component of microfibrils in the formation of elastic fibers in connective tissue Skeletal abnormality, subluxation of the lens (ectopia lentis), CVS abnormalities (mitral valve prolapse, aortic aneurysm, aortic dissection)
Examples
Skeletal: Marfan syndrome, Achondroplasia, Osteogenesis imperfecta Nervous system: Huntington disease, Neurofibromatosis Urinary: Polycystic kidney disease (Adult) Gastrointestinal: Familial polyposis coli Hematopoietic: Von Willebrand disease Metabolic: Familial hypercholesterolemia, Acute intermittent porphyria
http://www.uic.edu/classes/bms/bms655/lesson5.html
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Autosomal Recessive
Expressed only in homozygotes, otherwise can be trait There is horizontal inheritance (normal parents often have more than one affected child) Affected individuals have phenotypically normal parents Affected individuals usually have unaffected partners and all their children will be carriers
If a carrier has an unaffected partner, there is a 50% chance of the children being carriers Only mating between heterozygotes (carrier) will produce affected individuals, with an expected frequency of 1 in 4 There is an association with consanguinity due to sharing of genes in families (rare recessive genetic disorders are more likely to arise through consanguinity) Both sexes are equally affected and equally likely to pass the mutation to the next generation
http://upload.wikimedia.org/wikipedia/commons/thumb/3/3e/Autorecessive.svg/300px-Autorecessive.svg.png
http://healthnet.md.chula.ac.th/text/forum1/thalassemia2/picture/thalassemia.jpg
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Thalassemia Hemoglobinopathy Hemoglobin (Hb): iron-containing oxygen transport protein in the red blood cells Four globular protein subunits – Heme group containing Fe Two alpha(α) and two beta(β) subunits: [Heterotetramer; α2β2 = Hemoglobin A] Alpha thalassemia: HBA1 and HBA2 (16p13.3) Beta thalassemia: HBB (11p15.5)
ควรมีรูป Hb
http://gassama.myweb.uga.edu/hemoglobinmolecule.gif
Hemoglobinopathy Alpha thalassemia (decreased α chain) α chain mutation (4 loci) αα/αα, -α/αα, -α/-α, --/αα, --/-α, --/--
Beta thalassemia (decreased β chain) β chain mutation (2 loci) β/β, β/β0 , β/β+ , β+β+ , β0/β+ , β0/β0
Sickle-cell anemia β globin chain point mutation [missense mutation, Glu -> Val] Hemoglobin S (HbS) α2βS
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http://home.kku.ac.th/acamed/kanchana/p7.jpg
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http://www.microscopyu.com/galleries/pathology/images/sicklecell/sicklecellanemia40x02small.jpg
http://www.irondisorders.org/News/SickleCell.jpg
Example
Metabolic: Cystic fibrosis, Phenylketonuria, Hemochromatosis, Glycogen storage diseases, Galactosemia, Homocystinuria, Lysosomal storage diseases Hematopoietic: Thalassemia, Sickle cell disease Endocrine: Congenital adrenal hyperplasia Nervous: Friedreich ataxia
http://www.uic.edu/classes/bms/bms655/lesson7.html#RECESSIVE
X-linked Recessive
Many more males than females show the recessive phenotype The disease is transmitted by a carrier female, who is usually asymptomatic If a mother is a carrier, her son have a 50% chance of being affected and her daughters a 50% chance of being carriers An affected male will usually have no affected offspring, but all his daughters will be carriers and, in turn, 50% of their sons will be affected No sons of the affected male will inherit the gene (there is no male-to-male transmission)
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http://www.uic.edu/classes/bms/bms655/lesson7.html#RECESSIVE http://www.neurologyarticles.com/sitebuilder/images/Scan10106-176x315.jpg
Gower’s sign
http://medicalimages.allrefer.com/large/x-linked-recessive-genetic-defects.jpg
Duchenne muscular dystrophy
Example
Musculoskeletal: Duchenne muscular dystrophy, Becker muscular dystrophy Blood: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, Hemophilia A and B Metabolic: Diabetes insipidus, Lesch-Nyhan syndrome Nervous: Fragile-X syndrome (Atypical) Immune: Agammaglobulinemia
II. Chromosomal Abnormalities
Terminology Introduction Numerical Chromosome abn. Structural chromosome abn. Chromosome instability synd.
http://images.wikio.com/images/p/51ca/the-world-s-worst-genetic-mutation-in-a-dog.jpg
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Terminology Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another
http://www.transtutors.com/Uploadfile/CMS_Images/4026_Karyotype%20of%20human%20(Male).JPG
Terminology Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another
http://www.asklenore.info/miscarriage/bick/images/fig3.jpg
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Terminology Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another
http://www.ucl.ac.uk/~ucbhjow/bmsi/lec7_images/47_xx_21.gif
http://www.myoops.org/cocw/tufts/courses/20/content/D293242/C293223.jpg
Terminology Karyotype = the chromosome constitution of an individual, normal human karyotype is 46,XY (male) and 46,XX (female) Polyploidy = the number of hapoloid chromosome sets is greater than two (2n), triploidy, tetraploidy Aneuploidy = chromosome number is not an exact multiple of haploid number, monosomies, trisomies Trisomy = three representatives of a given chromosome, trisomy 21 in Down dyndrome Monosomy = one member of a chromosome pair is missing, Turner syndrome (45,X) Translocation = the transfer of one segment of a chromosome to another
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http://www.nature.com/onc/journal/v24/n33/images/1208711f1.jpg
Spectral Karyotyping (SKY) Analysis Introduction Maybe numerical or structural Nomenclature Numerical disorders : [47, XY,+21],
[45,X] Structural disorders : [46,XY,t(14;21)(q11,p10)],
[46,XY,dup(5) (q20-q30)], [46,XY,del(15)(q11-q13)],
(break point, margin, or region)
Numerical Chromosome Abnormalities
Concern : Extra single chromosome, Missing single chromosome, Extra haploid sets Mechanisms
Polyploidy Trisomies Monosomies
Polyploidy
Fertilization by two sperm A diploid sperm (2n) due to failure in meiosis A diploid ovum (2n) due to failure in meiosis
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Trisomies (aneuploidy) Failure of separation (nondisjunction) of homologous chromosomes at meiosis I Failure of separation of chromatids in meiosis II Advancing maternal age is associated with increased incidence of trisomy
http://www.anselm.edu/homepage/jpitocch/genbio/nondisjunction.JPG
Examples Autosomal disorders :
Trisomy 21 (Down syndrome) Trisomy 18 (Edwards syndrome) Trisomy 13 (Patau syndrome)
Sex chromosome disorders : Klinefelter syndrome (47,XXY) Turner syndrome (45,X)
XXX = female with an extra X chromosome XYY = male with an extra Y chromosome XX male = Y sequences are transferred to the X chromosome, look like male with Klinefelter syndrome
http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=108200&screenWidth=1280&screenHeight=800
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Down’s syndrome
CVS : Endocardial cushion defect (Atrioventricular septal defect) most common GI : increase risk of Hirschprung’s diseases
(intestinal stenosis) Down’s syndrome facies Simian crease Gap between 1st and 2nd toes
http://www.suriyothai.ac.th/en/node/1005
http://upload.wikimedia.org/wikipedia/commons/c/c3/Trisomy21_graph.jpg http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm
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http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=109400&screenWidth=1280&screenHeight=800 http://cas.bellarmine.edu/tietjen/HumanBioogy/bills_developmental_abnormalities.htm
Structural Chromosome Abnormalities
Result from breakage and limitations of DNA repair systems Mechanisms
Translocation Inversion Duplication Deletion and ring chromosome Isochromosome
http://www.robbinspathology.com/content/figure.cfm?ImageSeqNo=107050&screenWidth=1280&screenHeight=800
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http://cas.bellarmine.edu/tietjen/HumanBioogy/Finished%20Images/gen30.gif
mental retardation, low birth weight, low set ears, cat-like cry
Cri du chat syndrome (cat-like cry) : Deletion of the region on 5p15.2 or the whole short arm of chromosome 5
III. Multifactorial Disorders
Terminology Multifactorial disorders Threshold model of multifactorial disorders Examples of multifactorial disorders
Multifactorial Inheritance
Normal human characteristics : Blood pressure, height, finger ridges, and intelligence Congenital malformations : neural tube defects, cleft lip and palate, and congenital heart disease Common disorders of adult life : DM, Hypertension, peptic ulcer, and schizophrenia
Heritability and Environmental factors
Heritability : percentage denoting that the genetic contribution of a given disease
If heritability is high, there is a high correlation in relatives
Environmental factors : can be manipulated to reduce an individual’s susceptibility below than the threshold
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Threshold model for Multifactorial disorders
http://www.uic.edu/classes/bms/bms655/gfx/figure18.gif (Tendency)
Example
Diabetes mellitus, type I (Insulin-dependent) Essential hypertension (62% heritability) Atherosclerosis (65% heritability) Peptic ulcer (37% heritability) Schizophrenia (85% heritability) Asthma (80% heritability) Alzheimer disease
The age of expression of the major types of genetic disease Genetic disorders
Single gene disorders Mendelian inheritance
Autosomal dominant (AD) Autosomal recessive (AR) X-linked recessive (X-linked)
Non-Mendelian inheritance Chromosomal disorders
Numerical chromosomal abnormality Structural chromosomal abnormality
Polygenic or multifactorial diseases or complex genetic disorders Somatic cell genetic disorders (neoplasia)
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http://images.google.co.th/imgres?imgurl=http://www.web-books.com/eLibrary/Books/B0/B10/OPS/images/image023.jpg&imgrefurl=http://www.web-books.com/eLibrary/ON/B0/B10/23MB10.html&usg=__BPWTVs-v3b_lv3aPmVYlIh6BMlQ=&h=427&w=570&sz=26&hl=th&start=7&um=1&tbnid=XfWmaKv8PLBt0M:&tbnh=100&tbnw=134&prev=/images%3Fq%3Dtranslocation%2Bin%2Bleukemia%26ndsp%3D18%26hl%3Dth%26um%3D1
Molecular Diagnosis Techniques DNA , RNA Extraction Polymerase Chain Reaction (PCR) Gel Electrophoresis Direct Hybridization Method
Southern blot analysis Northern blot analysis
Fluorescence In situ Hybridization (FISH) DNA Microarrays Cytogenetics
Karyotyping Spectral karyotyping (SKY analysis)
http://www.favorgen.us/sitebuilder/images/Blood_Genomic_DNA_Mini_chart-437x463.jpg
DNA Extraction
Polymerase Chain Reaction (PCR)
http://www.obstbau-kompetenzzentrum.de/arbeitsbereiche/Folder.2006-11-07.1358/Thermocycler_.jpg http://www.bio.miami.edu/~cmallery/150/gene/c7.20.7.pcr.jpg
Taq DNA Polymerase
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http://users.ugent.be/~avierstr/principles/pcrcopies.gif http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate_600.jpg http://universe-review.ca/I11-50-gelectrophoresis.jpg
DNA Gel Electrophoresis
http://images.google.co.th/imgres?imgurl=http://users.ugent.be/~avierstr/principles/pcrcopies.gif&imgrefurl=http://users.ugent.be/~avierstr/principles/pcr.html&usg=__kCFieOdf4YXbyVmdNjY2uR9bquI=&h=306&w=598&sz=14&hl=th&start=9&um=1&tbnid=3Ku-pvmv7CJ2gM:&tbnh=69&tbnw=135&prev=/images%3Fq%3Dpcr%26ndsp%3D18%26hl%3Dth%26sa%3DN%26um%3D1
Southern Blot Analysis
http://www.mmi.mcgill.ca/mmimediasampler2002/images/5no15/5no15overview.gif
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Blot Detection Method Southern blot = DNA detection (DNA probe)
Edwin Southern, Professor of Biochemistry, 1975 Northern blot = RNA detection (RNA probe) Western blot = Protein detection (Antibody) Southwestern blot = DNA binding protein (DNA probe) Eastern blot = Protein post translation modification (probe detect CHO, lipid, etc.) Dot blot (Slot blot) = Nucleotide or Protein
Fluorescent In situ Hybridization
Localized specific DNA sequences on chromosomes using fluorescent probes Fluorescence microscopy Localized specific mRNA = gene expression
http://micro.magnet.fsu.edu/primer/techniques/fluorescence/anatomy/images/fluoromicrofigure1.jpg
http://en.wikipedia.org/wiki/File:FISH_(Fluorescent_In_Situ_Hybridization).jpg http://www.nature.com/modpathol/journal/v18/n12/images/3800465f1.jpg
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DNA Microarrays
http://img.medscape.com/fullsize/migrated/430/330/cc430330.fig2.gif http://en.wikipedia.org/wiki/File:NA_hybrid.svg http://en.wikipedia.org/wiki/File:Affymetrix-microarray.jpg
http://www.fao.org/DOCREP/003/X6884E/x6884e00.jpg http://neurophilosophy.files.wordpress.com/2006/08/cdna-array.jpg http://farm3.static.flickr.com/2077/2034113679_ac5a5c688d_m.jpg
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http://www.virtualsciencefair.org/2004/mcgo4s0/public_html/t2/dwn_chromo.gif
Cytogenetics : Karyotyping Spectral Karyotyping (SKY) Analysis
http://www.cityofhope.org/PublishingImages/Research/Shared%20Resources%20-%20Equipment/Cytogenetics%20Core%20Laboratory/HumanSKY_Large.jpg
References คัมภิรานนท, อมรา., “พันธุศาสตรมนุษย (Human Genetics), พิมพครั้งที่ 2.” เท็กซ แอนด เจอรนัล พับลิเคชั่น, กรุงเทพมหานคร, 2546.
บุญแสง, วิชัย., และคณะ., “ลายพิมพดีเอ็นเอ..จากสารพันธุกรรมสูเทคโนโลยีพิสูจนบุคคล, พิมพครั้งที่ 2.” สํานักงานพัฒนาวิทยาศาสตร และเทคโนโลยีแหงชาติ, ปทุมธานี, 2545.
Gelehrter, T.D., Collins, F.S., Ginsburg, D., “Principles of Medical Genetics, 2nd edition.” Williams & Wilkins, Maryland, 1998.
Lamb, N.E., “Crash course : Cell Biology and Genetics.” Mosby-Elsevier, Inc., Philadelphia, 2007.
Kumar, V., et al., “Robbins and Cotran Pathologic Basis of Disease, 7th edition.” Elsevier Inc., Philadelphia, 2005.
Rimoin, D. L., editors, et al., “Emery and Rimoin’s Principles and Practice of Medical Genetics, 4th edition.” Churchill Livingstone, London, 2002, volume 1.