Genetic Testing

Karyotyping
y yp g
Eva Diah Setijowati

Mazen Zaharna Molecular Biology 1/2009
Human Chromosomes
A normal
normal human carries 23 PAIRS of
chromosomes (1 set came from the
mother 1 set came from the father)
mother,
22 of these sets are called autosomes (or
self
self chromosomes)
chromosomes )
1 set are the sex chromosomes
A female carries two X chromosomes (XX)
A male carries an X chromosome and a Y
chromosome (XY)
Why do doctors look at

chromosomes?
h
?
To diagnose or predict genetic disorders
by looking at chromosomes.
Prenatal testing and in diagnosing certain
disorders
Down
D
syndrome,
d
or in diagnosing a specific types of leukemia.
Chromosome abnormalities
numerical
extra (47,XX,+21)
missing chromosomes (45
(45,X)
X)
structural
translocations
inversions
large scale deletions or duplications (>4Mbp)
Conditions where karyotyping is

strongly
l recommended
d d
Prenatal diagnosis
g
for fetus with family
y history
y of chromosome
aberration and previous child born with chromosome abnormality
Fertility problems
Neoplasia
Children with developmental delay
Children with mental retardation
Children
Child
with
ith multiple
lti l congenital
it l abnormalities
b
liti
Disorder Sexual Development (Sexual Ambiguity)
Short stature female
Couple with recurrent abortus history
Pregnancy in older women (>35
y.o)
What is a Karyotype?
A display or photomicrograph of
an individuals somatic-cell
metaphase chromosomes that
are arranged in a standard
sequence
Performing a Karyotype
The slides are scanned for metaphase spreads
and usually 20 to 100 (for mosaicism cases)
cells are analyzed under the microscope by a
cytogeneticist.
When a good spread (minimum number of
overlapping chromosomes) is found, a
photograph is taken or the analysis is done by a
computer.
computer
The chromosomes are arranged in a standard
presentation format
format.
Identify Chromosomes
Three key features to identify their

similarities
i il iti and
d diff
differences:
Size. This is the easiest way to tell
two different chromosomes apart.
p
Banding pattern. The size and
location of Giemsa bands on
chromosomes
h
make
k each
h
chromosome pair unique.
Centromere position. Centromeres
are regions in chromosomes that
appear as a constriction.
In metacentric chromosomes, the centromere lies near the

center of the chromosome.
Submetacentric & very Submetacentric chromosomes,
have a centromere that is off-center, so that one
chromosome arm is longer than the other
other.
In acrocentric chromosomes, the centromere resides very
near one end.
Chromosome banding
Chromosomes are stained with various
dyes enabling the chromosome segments
to be identified
Most methods can distinguish 550 bands/
haploid set
High resolution methods can distinguish
up to
t 850 bands/
b d /h
haploid
l id sett th
thatt can allow
ll
identification of small interstitial deletions
BANDING OF CHROMOSOMES
G - Banding
Q - Banding
g
C - Banding
R - Banding
g
T - Banding
NOR - Banding
High Resolution Banding
Restriction Endonuclease Banding
G Banding
G-Banding
Dye gives chromosomes a striped appearance
because it stains the regions of DNA that are rich in
adenine (A) and thymine (T) base pairs.
G Banding
G-Banding
Regions that stain as dark G bands
replicate late in S phase of the cell cycle
and contain more condensed chromatin
chromatin,
While light G bands generally replicate
early in S phase,
phase and have less
condensed chromatin.
Chromosomal Abnormalities
Alterations in chromosome number.

Euploid
E l id - normall sett (2
(2n))
Polyploidy extra set of the entire genome.
(3 4
(3n,
4n etc)
t )
Aneuploidy the number of chromosomes is

not a multiple of the normal haploid number
number.
Monosomy
one member of a chromosome p
pair is missing,
g, ((2n-1))
Trisomy
one chromosome set consists of 3 copies of a
chromosome (2n+1)
chromosome,
Chromosomal abnormalities that can

b d
be
detected
t t db
by kkaryotyping
t i
abnormal
Chromosomal abnormalities that can

b d
be
detected
t t db
by kkaryotyping
t i
Philadelphia Chromosome - CML
Overview of Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9
9.
Collection of blood
Cell culture
Stopping the cell division at Metaphase
Hypotonic treatment of red & white blood cells
Fixation
Slide preparation
Slide
S
de de
dehydration
yd a o
Treatment with enzyme
Staining
KULTUR SEL
5 ml PB Max dimasukkan dalam nunc flash

Tambahkan 0,3
, - 0,4
, ml darah
Campur dengan cara dibolak balik
Bersihkan nuncflash dengan kapas alkohol
Tutup nuncflash dikendorkan sebelum dimasukkan
dalam inkubator
PENAMBAHAN COLCEMID
30 menit sebelum harvest ambil tabung dari

inkubator
Tambahkan
T b hk C
Colcemid
l
id 0
0,2
2 mll campur d
dengan
cara dikocok pelan-pelan
Inkubasi lagi selama 30 menit
menit.
HARVESTING
72 JAM MENJELANG HARVESTING .
ENDAPAN YANG DIANALISA
21
22
Advantages and Disadvantages of

conventional cytogenetic technique
Advantages
g
1. Enable the entire genome to be viewed at one time.
2. Suitable when a specific anomaly is suspected ( e.g.
Philadelphia in CML ) and as a general diagnostic tool
to detect additional chr. Abnormalities commonly seen
in disease progression of CML.
Disadvantages
1. Detect major structural abnormalities
(one band = 6mb of DNA ~ 150 genes ).
)
2. Labor intensive and highly dependent upon operator

experience and skills.
Fluorescence in situ Hybridization
Fluorescence in situ Hybridization (FISH)
y FISH - a process which vividly
paints chromosomes or portions

of chromosomes with
fluorescent molecules
y Identifies chromosomal
abnormalities
y Gene mapping, analysis of
chromosome structural aberrations
aberrations,
and ploidy determination
y Used to identify the presence and
location of a region of DNA or RNA

within
ithi morphologically
h l i ll preserved
d
chromosome p
preparations,
p
, fixed
cells or tissue sections
y View a segment or entire
chromosome with your own eyes

y Was often used during M phase but
is now used on I phase
chromosomes as well
y Advantage: less labor-intensive

labor intensive
method for confirming the presence

off a DNA segmentt within
ithi an entire
ti
genome than other conventional
g
methods
Tissue samples
p for FISH analysis
y
y Peripheral blood
y Fibroblasts from skin biopsy
y Epithelial cells from buccal smear
y Bone
B
marrow (hemoblastosis)
(h
bl
i )
y Solid tumor biopsies
p
FISH Procedure
y Denature
D
t
th
the chromosomes
h
y Denature the probe
y Hybridization
y Fluorescence staining
y Examine
E
i slides
lid
FISH Procedure
FISH Uses
y Centromere regions stained
brighter - means they are rich in

A-T bonds
y Also used in germ cell or
prenatal diagnosis of conditions
such as aneuploidies
FISH and Telomeres

y Telomeric probes define the
terminal boundaries of
chromosomes (5 and 3 ends)
y Used in research of chromosomal
rearrangements and deletions
related to cell aging or other
genetic abnormalities
FISH and Telomeres

y Special telomeric probes
specific to individual
chromosomes have been
designed
y Probe is based on the TTAGGG
repeat present on all human
telomeres
FISH and Telomeres
FISH and Telomeres

y Application in cytogenetics - can
detect submicroscopic deletions

and cryptic translocations of genes
associated with unexplained mental
retardation and miscarriages
Green: Chromosome
G
Green:
Ch
13
Red:: Chromosome 21
Red
Normal
40
Down
syndrome
Buccal Cells from Swab
Dual fusion probes

y Sometimes called dual-colored
probes
y 0.81.5 Mb in size
y Designed
D i
d to
t bi
bind
d tto regions
i
spanning the breakpoint of both
translocation partners.
y A translocation will be observed
as a signal from both the
translocation junction and the
reciprocal of the translocation
junction; e.g., t(9;22) and t(22;9)
42
43
Limitations of FISH
y The inability to identify chromosomal changes other than
those
h
at the
h specific
ifi bi
binding
di region
i off the
h probe.
b
y Preparation of the sample is critical in interphase FISH
analysis
y
{
{
to permeabilize the cells for optimal probe target interaction

to maintain cell morphology.
y Cannot detect small mutations.

mutations
y Miss Uniparental disomy.
y Miss Inversions.
y Probes are not yet commercially available for all
chromosomal regions
y Relativelly expensive
44
Diagnostic Potential For Karyotype and FISH For

Selected Disorders
Condition
Locus
studied
Karyotype
Disease
specific FISH
Telomere FISH
Aneuploidy
various
~100%
Not detected
Detected by
karyotype
Large deletions, large

dupllications, translocation of
large segments
various
~100%
Not detected
Detected by
karyotype
Cryptic
Rearrangements of telomeres
various
Not
detected
Not detected
~100%
1 36 deletion
1p36
d l ti
1 36 3
1p36.3
F
Few
~99%
99%
>95%
95%
Wolf-Hirschhorn
4p16.3
Most
~99%
>95%
Cri-du-chat
5p15.2
Most
~99%
>95%
Willi
Williams-Beuren
B
7 11 2
7q11.2
Al
Almost
t none
~99%
99%
N t detected
Not
d t t d
Prader-Willi
15q11-q13
Unreliable
~70%
Not detected
Angelman
15q11-q13
Unreliable
~70%
Not detected
Mill Di k lissencephaly
Miller-Dieker
li
h l
17 13 3
17p13.3
F
Few
>90%
90%
Smith-Magenis
17p11.2
Some
>95%
Not detected
45
Velocardiofacial/DiGeorage
1
22q11.2
Rarely
>95%
Not detected
S
Some
d
detected
t t d
Polymerase Chain Reaction (PCR)

PolymeraseChainReaction(PCR)
PolymeraseChainReaction
Polymerase:
P l
DNA l
DNApolymerase
DNApolymeraseduplicatesDNA
Beforeacelldivides,itsDNAmustbe
duplicated
ChainReaction: Theproductofareactionis
usedtoamplifythesamereaction
dt
lif th
ti
Resultsinrapidincreaseintheproduct
ThePCRReactionComponents
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the
sequence to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA
building blocks.
4) Thermostable DNA Polymerase - enzyme that
catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution maintains pH and ionic strength of
th reaction
the
ti
solution
l ti suitable
it bl for
f the
th activity
ti it off the
th
enzyme
DNA polymerase
DNApolymerase
Duplicates
DuplicatesDNA
DNA
Necessaryforreproductionofnewcells
MorethanoneDNApolymerasesexistin
h
l
i i
differentorganisms
PropertiesofDNApolymearse
NeedsapreexistingDNAtoduplicate
Cannotassembleanewstrandfrom
components
CalledtemplateDNA
CanonlyextendanexistingpieceofDNA
Calledprimers
5
DNA
DNApolymeraseneedsMg
polymerase needs Mg++ ascofactor
as cofactor
EachDNApolymeraseworksbestunder
optimal temperature pH and salt
optimaltemperature,pHandsalt
concentration
PCRbufferprovidesoptimalpHandsalt
PCR b ff
id
i l H d l
condition
DNAstrandsareanti
DNA strands are antiparallel
parallel
Onestrandgoesin5 3
Thecomplementarystrandisopposite
The complementary strand is opposite
DNA
DNApolymerasealwaysmovesinone
l
l
i
direction(from5 3)
5
DNA
DNApolymeraseincorporatesthefour
polymerase incorporates the four
nucleotides(A,T,G,C)tothegrowingchain
dNTPfollowstandardbasepairingrule
dNTP follow standard base pairing rule
dTTP
dATP dATP
dATP
5 dGTP
dTTP
dTTP dCTP
dATP
dATP
dTTP
dGTP dGTP
dTTPdATP dCTP
dTTP
dGTP
dCTP
dGTP
dATP
dCTP
dCTP
dGTP
dCTP
The
ThenewlygeneratedDNAstrandsserveas
newly generated DNA strands serve as
templateDNAforthenextcycle
PCRisverysensitive
PCR is very sensitive
Widelyused
SettingupaPCRReaction
AddtemplateDNAandprimers
Add dNTPs
Add DNA polymerase

dATP dATP
dATP
dGTP
dTTP
dTTP dCTP
dATP
dATP
dTTP
dGTP dGTP
dTTPdATP dCTP
dTTP
dGTP
dTTP
dCTP
dGTP
dATP
dCTP
dCTP
dGTP
3
dCTP
Taq DNA polymerase

TaqDNApolymerase
DerivedfromThermusaquaticus
Derived from Thermus aquaticus
HeatstableDNApolymerase
Idealtemperature72C
d l
2C
Thermal Cycling
ThermalCycling
APCRmachinecontrolstemperature
p
TypicalPCRgothroughthreesteps
Denaturation
D t ti
Annealing
Extension
PCR tube
THERMOCYCLER
Overview of PCR
OverviewofPCR
1 Temperature Cycling
1.
Denaturation
A
Annealing
li
Extension
94
55
72
2. Every cycle DNA

between primers is
duplicated
Denaturation
Heatingseparatesthedouble
stranded DNA
strandedDNA
Denaturation
Slow
Slowcoolingannealsthetwo
cooling anneals the two
strands
Renaturation
Heat
Cool
A
Annealing
li
Twoprimersaresuppliedinmolarexcess
Theybindtothecomplementaryregion
AstheDNAcools,theywedgebetweentwo
As the DNA cools, they wedge between two
templatestrands
Optimaltemperaturevariesbasedon
Optimal temperature varies based on
primerlengthetc.
T i lt
Typicaltemperaturefrom40to60C
t
f
40 t 60 C
Extension
DNA polymerase duplicats DNA

Optimal temperature 72C
PCR Amplification
PCRAmplification
E
Exponential
ti l A
Amplification
lifi ti off ttemplate
l t DNA
Typical PCR mix

TypicalPCRmix
InathinwallEppendorftubeassemblethe
followingg
PCR components
Amount
Template DNA (5(5-200 ng

ng))
1 mM dNTPs (200 uM final)
10 X PCR buffer
25 mM
M MgCl2
M Cl2 (1.5
(1 5 mM
M final)
fi l)
20 uM forward primer (20 pmoles final)
20 uM reverse primer (20 pmoles final)
5 units/uL
units/uL Taq DNA polymerase (1.5 units)
Water
variable
10 uL
5 uL
3 uLL
1 uL
1 uL
0.3 uL
Variable
Final Volume
50 uL
Primers
PCRprimersareshort,singlestrandedDNA
p
g
molecules(1540bp)
Theyaremanufacturedcommerciallyandcanbe
ordered to match any DNA sequence
orderedtomatchanyDNAsequence
Primersaresequencespecific,theywillbindtoa
particularsequenceinagenome
Asyoudesignprimerswithalongerlength(1540
bp),theprimersbecomemoreselective.
DNApolymeraserequiresprimerstoinitiate
DNA polymerase requires primers to initiate
replication
Selectivity of Primers
SelectivityofPrimers
Primers
Primersbindtotheircomplementarysequenceon
bind to their complementary sequence on
thetargetDNA
Aprimercomposedofonly3letter,ACC,forexample,
wouldbeverylikelytoencounteritscomplementina
genome.
Asthesizeoftheprimerisincreased,thelikelihoodof,for
As the size of the primer is increased the likelihood of for
example,aprimersequenceof35baselettersrepeatedly
encounteringaperfectcomplementarysectiononthe
targetDNAbecomeremote.
Applications
ApplicationsofPCR
of PCR
Classification
of organisms
Genotyping
Molecular
archaeology
Mutagenesis
Mutation
detection
Sequencing
Cancer research
Detection of
th
pathogens
DNA
fingerprinting
Drug discovery
Genetic
matching
Genetic
engineering
Pre-natal
di
diagnosis
i
ApplicationsofPCR
Basic Research
Applied Research
Mutation screening
Drug discovery
Classification of organisms
G
t i
Genotyping
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
Bioinformatics
Genomic cloning
Site-directed mutagenesis
Gene
G
expression
i studies
t di
Genetic matching
Detection of pathogens
g
Pre-natal diagnosis
DNA fingerprinting
Gene therapy
ApplicationsofPCR
Molecular Identification Sequencing
Genetic Engineering
Site-directed mutagenesis
Molecular Archaeology Bioinformatics
Molecular Epidemiology Genomic cloning
Gene expression studies
Molecular
M l
l E
Ecology
l
Human Genome Project
DNA fingerprinting
Classification of organisms
Genotyping
Pre-natal
P
t l di
diagnosis
i
Mutation screening
Drug discovery
Genetic matching
Detection
D t ti off pathogens
th
Pulsed-Field Gel Electrophoresis (PFGE)
Repetitive Sequence-Based PCR

Random Amplified Polymorphic DNA (RAPD)
Amplified fragment length polymorphism
PCR and Disease

PCRandDisease
Primerscanbecreatedthatwillonlybindandamplify
certain alleles of genes or mutations of genes
certainallelesofgenesormutationsofgenes
ThisisthebasisofgeneticcounselingandPCRisusedaspart
ofthediagnostictestsforgeneticdiseases.
Some
Somediseasesthatcanbediagnosedwiththehelpof
diseases that can be diagnosed with the help of
PCR:(couldnotbedetectedbycytogenetictechniques)
Huntington'sdisease
A h d l i
Achondroplasia
Humanimmunodeficiencyvirus
Thalassemia
AvianInfluenzaVirus
DMD/BMD
etc
PCR and Forensic Science

PCRandForensicScience
Forensicscienceistheapplicationofabroadspectrumofsciencesto
answerquestionsofinteresttothelegalsystem.Thismaybeinrelationto
i
fi
h l l
Thi
b i
l i
acrimeortoacivilaction.
Itisoftenofinterestinforensicsciencetoidentifyindividualsgenetically.
It
is often of interest in forensic science to identify individuals genetically
Inthesecases,oneisinterestedinlookingatvariableregionsofthe
genomeasopposedtohighlyconservedgenes.
PCRcanbeusedtoamplifyhighlyvariableregionsofthehumangenome.
PCR
b
d
lif hi hl
i bl
i
f h h
Theseregionscontainrunsofshort,repeatedsequences(knownas
variable numberoftandemrepeat(VNTR)sequences).Thenumberof
repeatscanvaryfrom440indifferentindividuals.
Primersarechosenthatwillamplifytheserepeatedareasandthe
genomicfragmentsgeneratedgiveusauniquegeneticfingerprintthat
can be used to identify an individual.
canbeusedtoidentifyanindividual.
PCR Applications to Forensic Science

PCRApplicationstoForensicScience
PaternitysuitsArgentinasMothersoftheplazaand
theirsearchforabductedgrandchildren
Identifyingbadlydecomposedbodiesorwhenonly
b d f
bodyfragmentsarefound
f
d Worldtradecenter,Bosnian
W ld d
B i
,Iraq&Rwandanmassgraves
Crime Scene Investigator PCR

CrimeSceneInvestigatorPCR
IntroductiontoDNAprofiling
SetupPCRreactions
ElectrophoresisPCRproducts
Electrophoresis PCR products
Analysisandinterpretationofresults
DNAprofilingistheuseofmoleculargeneticmethodstodetermine
theexactgenotypeofaDNAsampleinawaythatcanbasically
distinguishonehumanbeingfromanother
TheuniquegenotypeofeachsampleiscalledaDNAprofile.
HowdocrimesceneinvestigatorscreateaDNAprofile?
g
p
1.Evidenceiscollectedatthecrimescene:
Blood
Tissue
Semen
Teeth
Urine
Hair
Saliva
Bone
How do crime scene investigators create a DNA profile?

2. DNA is extracted from sources at the crime
scene and from victim and suspects
How do crime scene investigators create a DNA

p
profile?
3. DNA samples are processed
Sample Obtained from
Crime Scene or
Paternity Investigation
DNA
Extraction
Biology
DNA
Quantitation
PCR Amplification
p
of multiple (STR) markers
Technology
Separation and Detection of PCR
Products
(STR Alleles)
Sample
S
l G
Genotype
t
Determination
Genetics
Comparison of Sample
Genotype to Other Sample
Results
Generation of Case Report

with Probability of Random
Match
If match occurs, comparison of
DNA profile to population
databases
Since humans are 99.9% identical

g
look
where do crime scene investigators
for differences in DNA profiles?
4. Crime Scene Investigators search in areas of
the genome that are unique from individual to
individual and are anonymous (control no known trait
or function)
f
) The areas examined are Short Tandem
Repeats or STRs
STR region
These STR sequences do NOT code for anything, i.e. they are NOT genes.
Example of an STR: TH01

The TH01 locus contains repeats of TCAT.
CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
This example has 6 TCAT repeats.
There are more than 20 known TH01 alleles.

alleles
Each individual inherits 1 allele from each parent.
Determining genotypes for individuals using STRs
Ms. Smiths TH01 locus for her two chromosomes

is given below.
What is her genotype?
MOMS CHROMOSOME
CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
DADS
DAD
S CHROMOSOME
CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT
TCAT TCAT TCAT TCAT TCAT AAA
Todeterminethegenotype(DNAprofile)CrimeSceneInvestigators
make billions of copies of the target sequence using PCR
makebillionsofcopiesofthetargetsequenceusingPCR
Target DNA
3
Starting DNA
Template
WhatssthepointofPCR?
What
the point of PCR?
PCR,orthepolymerasechainreaction,makes
copiesofaspecificpieceofDNA
PCRallowsyoutolookatonespecificpieceofDNA
b
bymakingcopiesof*only*thatpieceofDNA
ki
i
f * l * th t i
f DNA
PCRislikelookingforaneedleinahaystack,and
PCR
is like looking for a needle in a haystack and
thenmakingahaystackoutoftheneedle
DNA profiling is used to

determine which suspect
can not be excluded from
suspicion.
suspicion
Howaresuspectsincludedorexcludedfromaninvestigation?
Suspectsareincluded inaninvestigationiftheirDNA
profile matches withgenotypesfoundatthecrime
profilematches
with genotypes found at the crime
scene
SSuspectscanbeexcluded
t
b e cl ded iftheirDNAprofiledoesnot
if th i DNA
fil d
t
matchgenotypesfoundatthecrimescene
CrimeSceneInvestigatorPCRBasics ProceduresOverview
To visualize PCR products Crime Scene investigators use gel electrophoresis
TH01
alleles
(14)
(13)
(12)
( )
(11)
(10)
(9)
(8)
(7)
( )
(6)
(5)
(4)
(3)
Allele
ladder
Mother
Father
Child C
Child D
Child E
1 The mother,
1.
mother
father and child
all have saliva
taken from their
mouth.
2. This gets sent to a

laboratory where DNA
profiling
fili
ttakes
k place.
l
3. The results are studied and the parents informed.
Family
yA
What do the
results show?
Mother
Child
Father
Family
yB
What do the
results show?
Mother
Son
Daughter
Father
GeneticTesting
PredictivetestingTellsa
personifshecarriesamutation
, p
thatwillcause,orputherat
higherriskfor,adiseaselaterin
life.
N b
Newbornscreening
i
Detectscommondisordersin
newborns,whereimmediate
treatmentcanpreventdangerous
symptoms
Carriertesting
g
Tellsa
personwhetherornothecarries
amutationthatcouldbepassed
ontohisoffspring.Onecanbea
carrier,butnotbeatriskfora
disease(asinrecessivegenes)
(
g
)
GeneticTesting
PredictivetestingTellsa
personifshecarriesamutation
, p
thatwillcause,orputherat
higherriskfor,adiseaselaterin
life.
N b
Newbornscreening
i
Detectscommondisordersin
newborns,whereimmediate
treatmentcanpreventdangerous
symptoms
Carriertesting
g
Tellsa
personwhetherornothecarries
amutationthatcouldbepassed
ontohisoffspring.Onecanbea
carrier,butnotbeatriskfora
disease(asinrecessivegenes)
(
g
)
Physician
Genetic
Counselor
Test family
members with
di
disease
symptoms?
Test patient?
Reproduction
Prevention
Treatment
Physician
Genetic Counselor
GeneticTesting:
Genetic
Testing:
MolecularTechniques
DNA
RNA
Protein
Function
Genetic testing
DNA
RNA
Protein
Function
Levels of Genetic Testing

normal
mutated
Analysis of whole
chromosomes for large
changes; extra chromosome, very
l
large
d
deletions
l ti
or iinsertions
ti
DNA
atcgatcgatcg
atcgaAcgatcg
Analysis of protein shape for

any change that may affect the
folding of the protein
Protein
Protein
Function
Analysis
A
l i off sequence for
f smallll
changes; mutations in the
sequence, small deletions or
insertions
Analysis of protein function if

the functional protein is supposed
to make something, then some
tests can detect the presence or
absence of the product
Levels of Genetic Testing

normal
mutated
Analysis of whole
chromosomes for large
changes; extra chromosome, very
l
large
d
deletions
l ti
or iinsertions
ti
DNA
atcgatcgatcg
atcgaAcgatcg
Analysis of protein shape for

any change that may affect the
folding of the protein
Protein
Protein
Function
Analysis
A
l i off sequence for
f smallll
changes; mutations in the
sequence, small deletions or
insertions
Analysis of protein function if

the functional protein is supposed
to make something, then some
tests can detect the presence or
absence of the product
Examples of Mutations in the DNA Sequence
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg
g g gggggggggggg ggg g g g g g g g
g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgta
gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa
aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg
atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc
tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta
tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag
gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag
cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct
agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc
tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Normal
gg
g
gggggggacacagcgatctaatataaatctgataatcaaaggtttttttttttcagctagcttacgatcgatctacgta
Single base pair mutation
(Sickle cell anemia)
gg
g
Deletion
(Cystic fibrosis)
gg
g
Deletion
(Duchenne muscular dystrophy)
gg
g
gggggggacacagcgatctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttttttcagctagct
tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgat
cgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaacc
atctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatc
gatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacg
atcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacg
agcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgt
ggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaa
aaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatc
tatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctag
gctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatcta
ggctatattcggatatc
Insertion
(Huntingtons disease)
gg
g
gggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgta
atatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctat
ctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggct
atattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacga
ggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacaca
gcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacg
tgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactag
agctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcg
gatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattc
ggatatc
Multiple mutations
(Diabetes, susceptibility to breast cancer)
gg
g
gggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgta
atatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctat
ctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggct
atattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacga
ggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacaca
gcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacg
tgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactag
agctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcg
gatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattc
ggatatc
Multiple mutations
(Diabetes, susceptibility to breast cancer)

Genetic Testing

Încărcat de

Informații document

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Genetic Testing

Încărcat de

Drepturi de autor:

Formate disponibile

Karyotyping

Eva Diah Setijowati

Why do doctors look at

Conditions where karyotyping is

Three key features to identify their

In metacentric chromosomes, the centromere lies near the

Mazen Zaharna Molecular Biology 1/2009

Alterations in chromosome number.

Aneuploidy the number of chromosomes is

Chromosomal abnormalities that can

Chromosomal abnormalities that can

Philadelphia Chromosome - CML

5 ml PB Max dimasukkan dalam nunc flash

30 menit sebelum harvest ambil tabung dari

72 JAM MENJELANG HARVESTING .

ENDAPAN YANG DIANALISA

Mazen Zaharna Molecular Biology 1/2009

Mazen Zaharna Molecular Biology 1/2009

Advantages and Disadvantages of

2. Labor intensive and highly dependent upon operator

Fluorescence in situ Hybridization

Fluorescence in situ Hybridization (FISH)

y FISH - a process which vividly

paints chromosomes or portions

Fluorescence in situ Hybridization (FISH)

Fluorescence in situ Hybridization (FISH)

y Used to identify the presence and

location of a region of DNA or RNA

Fluorescence in situ Hybridization (FISH)

y View a segment or entire

chromosome with your own eyes

Fluorescence in situ Hybridization (FISH)

y Advantage: less labor-intensive

method for confirming the presence

y Centromere regions stained

brighter - means they are rich in

FISH and Telomeres

FISH and Telomeres

FISH and Telomeres

FISH and Telomeres

detect submicroscopic deletions

Buccal Cells from Swab

Dual fusion probes

to permeabilize the cells for optimal probe target interaction

y Cannot detect small mutations.

Diagnostic Potential For Karyotype and FISH For

Large deletions, large

Polymerase Chain Reaction (PCR)

Add DNA polymerase

Taq DNA polymerase

2. Every cycle DNA

DNA polymerase duplicats DNA

Typical PCR mix

Template DNA (5(5-200 ng

Pulsed-Field Gel Electrophoresis (PFGE)

Repetitive Sequence-Based PCR

PCR and Disease

PCR and Forensic Science

PCR Applications to Forensic Science

Crime Scene Investigator PCR

How do crime scene investigators create a DNA profile?

How do crime scene investigators create a DNA

Generation of Case Report

Since humans are 99.9% identical