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
ƒ Prenatal diagnosis
g for fetus with family
y history y of chromosome
aberration and previous child born with chromosome abnormality
ƒ Fertility problems
ƒ Neoplasia
ƒ Children with developmental delay
ƒ Children with mental retardation
ƒ Children
Child with
ith multiple
lti l congenital
it l abnormalities
b liti
ƒ Disorder Sexual Development (Sexual Ambiguity)
ƒ Short stature female
ƒ Couple with recurrent abortus history

ƒ Pregnancy in older women (>35 y.o)

 What is a Karyotype?

ƒ A display or photomicrograph of
an individual’s 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:
ƒ Size. This is the easiest way to tell
two different chromosomes apart.
p
ƒ Banding pattern. The size and
location of Giemsa bands on
chromosomes
h make
k each h
chromosome pair unique.
ƒ 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 0.8–1.5 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)
Polymerase Chain Reaction (PCR)
 Polymerase Chain Reaction

• Polymerase:
P l DNA l
DNA polymerase
– DNA polymerase duplicates DNA
– Before a cell divides, its DNA must be 
duplicated

– Chain Reaction: The product of a reaction is 
used to amplify the same reaction 
dt lif th ti
– Results in rapid increase in the product
 The PCR “Reaction” Components 

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
DNA polymerase
• Duplicates
Duplicates DNA
DNA
• Necessary for reproduction of new cells
• More than one DNA polymerases exist in 
h l i i
different organisms
 Properties of DNA polymearse 

• Needs a pre‐existing DNA to duplicate
– Cannot assemble a new strand from 
components
– Called template DNA
• Can only extend an existing piece of DNA
– Called primers

5’ 3’

3’ 5’
 Properties of DNA polymearse 

• DNA polymerase needs Mg++ as cofactor

DNA polymerase needs Mg as cofactor
• Each DNA polymerase works best under 
optimal temperature pH and salt
optimal temperature, pH and salt 
concentration
• PCR buffer provides optimal pH and salt 
PCR b ff id i l H d l
condition
 Properties of DNA polymearse 

• DNA strands are anti
DNA strands are anti‐parallel
parallel
– One strand goes in 5’ Æ 3’
– The complementary strand is opposite
The complementary strand is opposite

• DNA
DNA polymerase always moves in one 
l l i
direction (from 5’ Æ 3’)
5’ 3’

3’ 5’
 Properties of DNA polymearse 

• DNA
DNA polymerase incorporates the four 
polymerase incorporates the four
nucleotides (A, T, G, C) to the growing chain
• dNTP follow standard base pairing rule
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’
 Properties of DNA polymearse 

• The
The newly generated DNA strands serve as 
newly generated DNA strands serve as
template DNA for the next cycle
• PCR is very sensitive
PCR is very sensitive
• Widely used
 Setting up a PCR Reaction 

• Add template DNA and primers
„ 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
Taq DNA polymerase
• Derived from Thermus aquaticus
Derived from Thermus aquaticus
• Heat stable DNA polymerase
• Ideal temperature 72C
d l 2C
 Thermal Cycling
Thermal Cycling
• A PCR machine controls temperature
p
• Typical PCR go through three steps
– Denaturation
D t ti
– Annealing
– Extension

PCR tube THERMOCYCLER

 Overview of PCR
Overview of PCR
1 Temperature Cycling
1.

Denaturation 94°
A
Annealing
li 55°
Extension 72°

2. Every cycle DNA

between primers is
duplicated
 Denaturation

• Heating separates the double 
stranded DNA
stranded DNA
– Denaturation
Heat Cool
• Slow
Slow cooling anneals the two 
cooling anneals the two
strands
– Renaturation
 A
Annealing
li

• Two primers are supplied in molar excess 
• They bind to the complementary region
• As the DNA cools, they wedge between two 
As the DNA cools, they wedge between two
template strands
• Optimal temperature varies based on 
Optimal temperature varies based on
primer length etc. 
T i lt
Typical temperature from 40 to 60 C
t f 40 t 60 C
 Extension

„ DNA polymerase duplicats DNA

„ Optimal temperature 72C
 PCR Amplification
PCR Amplification

E
Exponential
ti l A
Amplification
lifi ti off ttemplate
l t DNA
 Typical PCR mix
Typical PCR mix
In a thin wall Eppendorf tube assemble the 
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
• PCR primers are short, single stranded DNA 
p g
molecules (15‐40 bp)
• They are manufactured commercially and can be 
ordered to match any DNA sequence
ordered to match any DNA sequence
• Primers are sequence specific, they will bind to a 
particular sequence in a genome
• As you design primers with a longer length (15 → 40 
bp), the primers become more selective. 
• DNA polymerase requires primers to initiate 
DNA polymerase requires primers to initiate
replication
 Selectivity of Primers
Selectivity of Primers
• Primers
Primers bind to their complementary sequence on 
bind to their complementary sequence on
the target DNA
– A primer composed of only 3 letter, ACC, for example, 
would be very likely to encounter its complement in a 
genome. 
– As the size of the primer is increased, the likelihood of, for 
As the size of the primer is increased the likelihood of for
example, a primer sequence of 35 base letters repeatedly 
encountering a perfect complementary section on the 
target DNA become remote.
 Applications
Applications of PCR
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
 Applications of PCR
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
 Applications of PCR

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
PCR and Disease
• Primers can be created that will only bind and amplify 
certain alleles of genes or mutations of genes
certain alleles of genes or mutations of genes
• This is the basis of genetic counseling and PCR is used as part 
of the diagnostic tests for genetic diseases.
• Some
Some diseases that can be diagnosed with the help of 
diseases that can be diagnosed with the help of
PCR:  (could not be detected by cytogenetic techniques)
• Huntington's disease
• A h d l i
Achondroplasia
• Human immunodeficiency virus
• Thalassemia
• Avian Influenza Virus
• DMD / BMD
• etc
 PCR and Forensic Science
PCR and Forensic Science
• Forensic science is the application of a broad spectrum of sciences to 
answer questions of interest to the legal system. This may be in relation to 
i fi h l l Thi b i l i
a crime or to a civil action. 

• It is often of interest in forensic science to identify individuals genetically.  
It is often of interest in forensic science to identify individuals genetically
In these cases, one is interested in looking at variable regions of the 
genome as opposed to highly‐conserved genes. 

• PCR can be used to amplify highly variable regions of the human genome. 
PCR b d lif hi hl i bl i f h h
These regions contain runs of short, repeated sequences (known as 
variable number of tandem repeat (VNTR) sequences) .  The number of 
repeats can vary from 4‐40 in different individuals. 

• Primers are chosen that will amplify these repeated areas and the  
genomic fragments generated give us a unique “genetic fingerprint” that 
can be used to identify an individual.
can be used to identify an individual.
PCR Applications to Forensic Science
PCR Applications to Forensic Science

• Paternity suits ‐Argentina’s Mothers of the plaza and 
their search for abducted grandchildren

• Identifying badly decomposed bodies or when only 
b d f
body fragments are found ‐
f d World trade center, Bosnian 
W ld d B i
, Iraq & Rwandan mass graves
 Crime Scene Investigator PCR
Crime Scene Investigator PCR
• Introduction to DNA profiling

• Set up PCR reactions

• Electrophoresis PCR products
Electrophoresis PCR products

• Analysis and interpretation of results

• DNA profiling is the use of molecular genetic methods to determine 
the exact genotype of a DNA sample in a way that can basically 
distinguish one human being from another 

• The unique genotype of each sample is called a DNA profile.
How do crime scene investigators create a DNA profile? 
g p

1. Evidence is collected at the crime scene:

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 STR’s

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. Smith’s TH01 locus for her two chromosomes

is given below.

What is her genotype?

MOM’S CHROMOSOME

CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA

DAD’S
DAD S CHROMOSOME

CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT
TCAT TCAT TCAT TCAT TCAT AAA
To determine the genotype (DNA profile) Crime Scene Investigators 
make billions of copies of the target sequence using PCR
make billions of copies of the target sequence using PCR

Target DNA

5’ 3’
Starting DNA
Template
3’ 5’
What’ss the point of PCR?
What the point of PCR?
• PCR, or the polymerase chain reaction, makes 
copies of a specific piece of DNA

• PCR allows you to look at one specific piece of DNA 
b
by making copies of *only* that piece of DNA
ki i f * l * th t i f DNA

• PCR is like looking for a needle in a haystack, and 
PCR is like looking for a needle in a haystack and
then making a haystack out of the needle
DNA profiling is used to
determine which suspect
can not be excluded from
suspicion.
suspicion
How are suspects included or excluded from an investigation?

• Suspects are included in an investigation if their DNA 
profile matches with genotypes found at the crime 
profile matches with genotypes found at the crime
scene

• SSuspects can be excluded
t b e cl ded if their DNA profile does not 
if th i DNA fil d t
match genotypes found at the crime scene
Crime Scene Investigator PCR Basics Procedures Overview
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

 Genetic Testing
Predictive testing  Tells a 
person if she carries a mutation 
, p
that will cause, or put her at 
higher risk for, a disease later in 
life. ? ? ? ?

N b
Newborn screening 
i
Detects common disorders in 
newborns, where immediate 
treatment can prevent dangerous 
symptoms

Carrier testing       
g Tells a 
person whether or not he carries 
a mutation that could be passed 
on to his offspring.  One can be a 
carrier, but not be at risk for a 
disease (as in recessive genes)
( g )
 Genetic Testing
Predictive testing  Tells a 
person if she carries a mutation 
, p
that will cause, or put her at 
higher risk for, a disease later in 
life. ? ? ? ?

N b
Newborn screening 
i
Detects common disorders in 
newborns, where immediate 
treatment can prevent dangerous 
symptoms

Carrier testing       
g Tells a 
person whether or not he carries 
a mutation that could be passed 
on to his offspring.  One can be a 
carrier, but not be at risk for a 
disease (as in recessive genes)
( g )
Physician

Genetic
Counselor

?
Test family
members with
di
disease
symptoms?

Test patient?
 Reproduction
? Prevention

Treatment

Physician
Genetic Counselor
 Genetic Testing:
Genetic Testing:
Molecular Techniques
 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

(Huntington’s 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)