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.

Mazen Zaharna Molecular Biology 1/2009

 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
C - Bandingg
R - Banding
T - Banding g
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. Collection of blood
2. Cell culture
3. Stopping the cell division at Metaphase
4. Hypotonic treatment of red & white blood cells
5. Fixation
6. Slide preparation
7. Slide
S de de
dehydration
yd a o
8. Treatment with enzyme
9
9. 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 x y
Mazen Zaharna Molecular Biology 1/2009
Mazen Zaharna Molecular Biology 1/2009
Mazen Zaharna Molecular Biology 1/2009
 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
 Fluorescence in situ Hybridization (FISH)

y Identifies chromosomal
abnormalities
y Gene mapping, analysis of
chromosome structural aberrations
aberrations,
and ploidy determination
 Fluorescence in situ Hybridization (FISH)

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
 Fluorescence in situ Hybridization (FISH)

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
 Fluorescence in situ Hybridization (FISH)

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 Down
syndrome

40
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
bindd 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 Karyotype Disease Telomere FISH

studied specific FISH
Aneuploidy various ~100% Not detected Detected by
karyotype
Large deletions, large various ~100% Not detected Detected by
dupllications, translocation of karyotype
large segments
Cryptic various Not Not detected ~100%
Rearrangements of telomeres detected
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% S
Some d
detected
t t d
Smith-Magenis 17p11.2 Some >95% Not detected
Velocardiofacial/DiGeorage
45 1 22q11.2 Rarely >95% Not detected
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
 Discovery
PCRwasdiscoveredbyKaryMullis
PCR was discovered by Kary Mullis
Onalongmotorcycledrive
Mentallyvisualizedtheprocess
Mentally visualized the process

NobelPrizeinChemistry
N b l P i i Ch i t
1993
 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 3

3 5
 PropertiesofDNApolymearse

DNA polymerase needs Mg++ ascofactor

DNApolymeraseneedsMg as cofactor
EachDNApolymeraseworksbestunder
optimal temperature pH and salt
optimaltemperature,pHandsalt
concentration
PCRbufferprovidesoptimalpHandsalt
PCR b ff id i l H d l
condition
 PropertiesofDNApolymearse

DNAstrandsareanti
DNA strands are antiparallel
parallel
Onestrandgoesin5 3
Thecomplementarystrandisopposite
The complementary strand is opposite

DNA
DNApolymerasealwaysmovesinone
l l i
direction(from5 3)
5 3

3 5
 PropertiesofDNApolymearse

DNA
DNApolymeraseincorporatesthefour
polymerase incorporates the four
nucleotides(A,T,G,C)tothegrowingchain
dNTPfollowstandardbasepairingrule
dNTP follow standard base pairing rule

dTTP

dATP dATP dCTP dCTP

dATP dTTP dTTP dCTP dGTP
dATP
5 dGTP dATP 3
dTTP dGTP dGTP dATP dCTP

dTTPdATP dCTP dTTP dGTP dGTP dCTP

3 5
 PropertiesofDNApolymearse

The
ThenewlygeneratedDNAstrandsserveas
newly generated DNA strands serve as
templateDNAforthenextcycle
PCRisverysensitive
PCR is very sensitive
Widelyused
 SettingupaPCRReaction

AddtemplateDNAandprimers
Add dNTPs
Add DNA polymerase dTTP

dATP dATP dCTP dCTP

5 dATP dTTP dTTP dCTP dGTP
3
dATP
dATP
dGTP dTTP dGTP dGTP dATP dCTP

3 dTTPdATP dCTP dTTP dGTP dGTP dCTP 5

 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 94
A
Annealing
li 55
Extension 72

2. Every cycle DNA

between primers is
duplicated
 Denaturation

Heatingseparatesthedouble
stranded DNA
strandedDNA
Denaturation
Heat Cool
Slow
Slowcoolingannealsthetwo
cooling anneals the two
strands
Renaturation
 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)) variable
1 mM dNTPs (200 uM final) 10 uL
10 X PCR buffer 5 uL
25 mMM MgCl2
M Cl2 (1.5
(1 5 mMM final)
fi l) 3 uLL
20 uM forward primer (20 pmoles final) 1 uL
20 uM reverse primer (20 pmoles final) 1 uL
5 units/uL
units/uL Taq DNA polymerase (1.5 units) 0.3 uL
Water 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 Detection of
of organisms th
pathogens
Genotyping DNA
Molecular fingerprinting
archaeology Drug discovery
Mutagenesis Genetic
Mutation matching
detection Genetic
Sequencing engineering
Cancer research Pre-natal
di
diagnosis
i
 ApplicationsofPCR
Basic Research Applied Research

Mutation screening Genetic matching

Drug discovery Detection of pathogens
Classification of organisms g
Pre-natal diagnosis
Genotyping
G t i DNA fingerprinting
Molecular Archaeology Gene therapy
Molecular Epidemiology
Molecular Ecology
Bioinformatics
Genomic cloning
Site-directed mutagenesis
Gene
G expression
i studies
t di
 ApplicationsofPCR

Molecular Identification Sequencing Genetic Engineering

Molecular Archaeology Bioinformatics Site-directed mutagenesis
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
diagnosisi
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

Urine
Hair
Teeth 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 Biology

DNA DNA PCR Amplification

p
Extraction Quantitation of multiple (STR) markers

Technology

Separation and Detection of PCR Sample

S l G
Genotype
t
Products Determination
(STR Alleles)

Genetics
Comparison of Sample Generation of Case Report
Genotype to Other Sample with Probability of Random
Results Match

If match occurs, comparison of

DNA profile to population
databases
 Since humans are 99.9% identical
g
where do crime scene investigators look
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
) The areas examined are Short Tandem
or function)
f
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

5 3
Starting DNA
Template
3 5
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 Allele
Mother Father Child C Child D Child E
alleles ladder

(14)

(13)

(12)
( )
(11)

(10)

(9)

(8)

(7)

( )
(6)

(5)
(4)

(3)
 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
DNA chromosomes for large
changes; extra chromosome, very
l
large d
deletions
l ti or iinsertions
ti

Analysis
A l i off sequence for
f smallll
atcgatcgatcg atcgaAcgatcg
changes; mutations in the
sequence, small deletions or
insertions

Analysis of protein shape for

Protein any change that may affect the
folding of the protein

Analysis of protein function if

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

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

Analysis
A l i off sequence for
f smallll
atcgatcgatcg atcgaAcgatcg
changes; mutations in the
sequence, small deletions or
insertions

Analysis of protein shape for

Protein any change that may affect the
folding of the protein

Analysis of protein function if

Protein the functional protein is supposed
Function X X to make something, then some
tests can detect the presence or
absence of the product
Examples of Mutations in the DNA Sequence
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
Examples of Mutations in the DNA Sequence
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg g g gggggggggggg ggg g g g g g g g g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgta
gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa
aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg
atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc
tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta
tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag
gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag
cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct
agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc
tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Normal
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
Examples of Mutations in the DNA Sequence
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg g g gggggggggggg ggg g g g g g g g g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgataatcaaaggtttttttttttcagctagcttacgatcgatctacgta
gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa
aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg
atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc
tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta
tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag
gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag
cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct
agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc
tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Single base pair mutation

(Sickle cell anemia)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
Examples of Mutations in the DNA Sequence
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg g g gggggggggggg ggg g g g g g g g g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgta
gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa
aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg
atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc
tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta
tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag
gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag
cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct
agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc
tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Deletion

(Cystic fibrosis)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
Examples of Mutations in the DNA Sequence
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg g g gggggggggggg ggg g g g g g g g g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgta
gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa
aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg
atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc
tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta
tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag
gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag
cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct
agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc
tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc

Deletion

(Duchenne muscular dystrophy)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
Examples of Mutations in the DNA Sequence
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg g g gggggggggggg ggg g g g g g g g g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttttttcagctagct
tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgat
cgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaacc
atctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatc
gatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacg
atcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacg
agcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgt
ggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaa
aaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatc
tatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctag
gctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatcta
ggctatattcggatatc
Insertion

(Huntingtons disease)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
Examples of Mutations in the DNA Sequence
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg g g gggggggggggg ggg g g g g g g g g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgta
gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa
aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg
atatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctat
ctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggct
atattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacga
ggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacaca
gcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacg
tgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactag
agctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcg
gatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattc
ggatatc
Multiple mutations

(Diabetes, susceptibility to breast cancer)

aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
Examples of Mutations in the DNA Sequence
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct
atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta
ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa
aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc
gg g g gggggggggggg ggg g g g g g g g g
ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga
gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg
gggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgta
gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa
aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg
atatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctat
ctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggct
atattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacga
ggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacaca
gcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacg
tgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactag
agctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcg
gatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattc
ggatatc
Multiple mutations

(Diabetes, susceptibility to breast cancer)