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Genetic Diagnostic Method

Genetic disease and testing

• The patient’s journey…

• Types of genetic tests

• Techniques used in genetic testing

• Implications of genetic testing

• The future…

Inherited disease

Inherited disease

x23 x23

x23 x23 x23 x23 x23 x23

x23 x23 x23 x23 x23 x23

x23 x23 x23 x23 x23 x23

Genetic disease

Symptoms

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

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

Carrier testing 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)

? ? ? ?

Genetic Testing

?

Physician

Genetic

Counselor

Test family

members with

disease

symptoms?

Test patient?

?

Treatment

Prevention

Reproduction

Physician

Genetic Counselor

Genetic

Counseling

Dr. M. Houshmand

Dr. M. Houshmand

Diagnostic

We now know how God

wrote the book of life

But do we know

How to read the book ?

Genetic Diagnostics

• Cytogenetic tests

• FISH

• Molecular tests

DNA

RNA

Protein

Function

DNA

RNA

Protein

Function

Genetic testing

DNA

Protein

Protein

Function

Levels of Genetic Testing

normal mutated

Analysis of whole

chromosomes – for large

changes; extra chromosome, very

large deletions or insertions

atcgatcgatcg atcgaAcgatcg Analysis of sequence – for small

changes; mutations in the

sequence, small deletions or

insertions

Analysis of protein shape – for

any change that may affect the

folding of the protein

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

X X

DNA

Protein

Protein

Function

normal mutated

Analysis of whole

chromosomes – for large

changes; extra chromosome, very

large deletions or insertions

atcgatcgatcg atcgaAcgatcg Analysis of sequence – for small

changes; mutations in the

sequence, small deletions or

insertions

Analysis of protein shape – for

any change that may affect the

folding of the protein

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

X X

Levels of Genetic Testing

Molecular Biology Overview

Cell Nucleus

Chromosome

Protein

Graphics courtesy of the National Human Genome Research Institute

Gene (DNA) Gene (mRNA), single strand

Cytogenetic

Microscope

Molecular Cytogenetic

Biomedical Genetics

Molecular Genetic

Country

Chromosomal Abnormalities

Chromosome

1. Cells (from blood, amniotic fluid, or chorionic villus) are grown in culture. Mitogens may be required: lymphocytes require phytohemagglutinin

2. Colcemid stops cells at metaphase.

3. Hypotonic shock ruptures RBCs, swells lymphocytes.

4. Cells are fixed in MeOH/HOAc.

5. Chromosomes are spread on a slide.

6. Trypsinization and staining with Giemsa reveals G-bands.

7. The chromosome spread is photographed and arranged by type.

Karyotypes are made from metaphase chromosomes:

normal meiotic

division and

fertilization

Autosome

Comparative Scale of

Mapping

Country

~ 1 Week

Chromosomes Preparation

• Any tissue with living nucleated cells which undergo division can be used for studying chromosomes.

• Most commonly circulating lymphocytes from peripheral blood are used.

• Samples for chromosomal analysis can be prepared relatively easily using:

- Skin

- Bone marrow

- Chorionic Villi

- Amniocytes

G-banding analysis

Chromosomal Abnormalities

• Mutations when involve large parts of the

chromosome, and when these are large

enough to be visible under the light

microscope, they are termed

CHROMOSOME ABERRATION.

• With light microscope, the smallest visible

addition or deletion from a chromosome is

about 4 Mbp, which involves many

contiguous genes.

Chromosomal Aberrations

Abnormalities of the chromosomes are usually

classified into:

I. Numerical abnormalities: where the somatic cells contain an abnormal number of normal chromosomes.

II. Structural aberrations: where the somatic cells contain one or more abnormal chromosomes.

Common Aneuploidies

Trisomy 21 (Down Syndrome)

Trisomy 18 ( Edwards Syndrome)

Trisomy 13 ( Patau Syndrome)

Trisomy 16

47, XYY (Super males)

47, XXY (Klinefelter Syndrome)

47, XXX ( Super females)

45, X (Turner Syndrome)

Structural Aberrations

• Structural chromosome rearrangements result

from chromosome breakage with subsequent

reunion in a different configuration.

• They can be balanced or unbalanced.

Structural Aberrations

Structural aberrations are subdivided into:

• Translocations

• Deletions

• Duplications

• Inversions

• Ring chromosomes

• Isochromosomes

Country

State

~ 1 Week

+

Fluorescence In Situ Hybridization

“Conventional” Limitations

• Quality

– constitutional vs. acquired (oncology)

– ambiguous results (marker chromosomes)

• Time

– cell culture

– “tech time”

• Mitotic Images

– culture failures

– bias

Mosaic +18 (13, 18, 21, X, Y)

Multiplex-FISH

~ 1 Week

Comparative Genomic Hybridization

+

Normal donor

Array Comparative Genomic Hybridization

(aCGH)

Whole Genome Data Is Acquired

Patient below without any known genetic disease

All chromosomes but Y represented

Xp21 Complex Glycerol Kinase Deficiency

Log

2-r

atio

X chromosome

6.66 Mb deletion

Start:25274549

End:31940984

ACMG Recommends Replacing Karyotyping with

Chromosomal Microarrays as 'First-Line' Postnatal

Test

Microarrays should be used instead of G-banded

karyotyping as the first test to detect genetic

abnormalities in postnatal evaluations, according to

the American College of Medical Genetics.

September 28, 2010

CNVs are common in all genomes surveyed …

• Blue = pathogenic

• Red = deletion

• Green = duplication

CGH Breast Cancer

~ 2 days

DNA Micro Arrays (FISH & Chips )

+ Normal donor

Manufactured DNA chip

Gene loss

Gene gain

“Chip” Technology

District

People

Normal, wild type Mutation inactivates enzyme

Normal

Nonsense Mutation

Missense Mutation

Framshif Mutation

Mutation

Detections

Techniques

1993

http://bldg6.arsusda.gov/pberkum/Public/sarl/cregan/pcr.gif

Components of a PCR

Reaction

• Buffer (containing Mg++)

• Template DNA

• 2 Primers that flank the fragment of DNA to be amplified

• dNTPs

• Taq DNA Polymerase (or another thermally stable DNA polymerase)

PCR Melting

94 oC

Melting

94 oC

Annealing

Primers

50 oC

Extension

72 oC T

emp

erat

ure

100

0

50

T i m e

30x

5’ 3’

3’ 5’

3’ 5’

5’

5’ 3’ 5’

3’ 5’

5’

5’

5’

5’ 3’

3’ 5’

3’ 5’

5’ 3’

5’ 3’

5’

How do we “look”

at the DNA sequence?

• Sequencing

• RFLP analysis

• Probes

– Southern blot

– Dot blot

– Microarray

•Gel electrophoresis

•PCR

PCR (Polymerase Chain

Reaction)

aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta

PCR

aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat

ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta

Melting

94°C

PCR



Annealing

58°C

cgggcat

aactagg

PCR



Elongation

72°C

cgggcat

aactagg aatcgaatgtgcccgtacgattcgatgcga

gctaagctacgctttgatcctcgggatagcta

PCR



Melting

94°C

cgggcat



PCR



Annealing

58°C

cgggcat



cgggcat

cgggcat

aactagg

aactagg

PCR



Elongation

72°C

cgggcat



cgggcat

cgggcat

aactagg

aactagg

gcccgtacgattcgatgcga


aatcgaatgtgcccgtacgattcgatgcga

gctaagctacgctttgatcc

PCR Makes a lot of copies of a specific portion of DNA

PCR test for deletion

Electrophoresis

DNA Between The Primers Doubles

With Each Thermal Cycle

0

Cycles

Number

1

3

8

2

4

1

2

4

16

5

32

6

64

More Cycles = More DNA Number of cycles

0 10 15 20 25 30

Size

Marker

Theoretical Yield Of PCR Theoretical yield = 2n x y

Where y = the starting

number of copies and

n = the number of thermal cycles

= 107,374,182,400

If you start with 100 copies, how many copies are

made in 30 cycles?

2n x y

= 230 x 100

= 1,073,741,824 x 100

Gel Electrophoresis Separates DNA fragments on the basis of size

Large fragments take longer than

small fragments to migrate

through an agarose gel

DMD Multiplex deletion test

affected range: >39 repeats

CAG repeats in Huntington’s disease

normal range: 11-35 repeats

may or may not have disease: 36-39 repeats

normal but can expand: 27-35 repeats

(CAG)n

(Gln)n

Structure and inheritance of CAG repeats

in spinal and bulbar muscular atrophy

normal 13-28

affected 39-60

(CAG)n

(Gln)n

androgen receptor gene

Spinal Bulbar Muscular Atrophy

Kennedy Syndrome

Dr. Babak Zamani

Cancer

DMD

Paternity Testing

Forensic testing

Steps in Forensic DNA Analysis

DNA

Extraction

Multiplex PCR Amplification

Male: 13,14-15,16-12,13-10,13-15,16

Interpretation of Results

Sample Collection

& Storage

Buccal swab Blood Stain

DNA

Quantitation

Slot Blot 1 ng

0.3 ng

1 ng

1 ng

0.7 ng

0.5 ng

0.5 ng

No DNA

Usually 1-2 day process (a minimum of ~5 hours)

If a match occurs, comparison of

DNA profile to population allele

frequencies to generate a case

report with probability of a random

match to an unrelated individual

STR Typing

DNA separation and sizing

Tec

hn

olo

gy

B

iolo

gy

Ge

ne

tic

s

DNA

Database

Search

Collection

Extraction

Quantitation

STR Typing

Interpretation

of Results

Database Storage & Searching

Specimen Storage

Multiplex PCR

Calculation of

Match Probability

Steps Involved

Short Tandem Repeat (STR) Markers

STR repeat region

GATA GATA GATA GATA

PCR product size generated

DNA template

containing STR marker

Reverse

PCR primer

Forward

PCR primer

Fluorescent

dye

PCR Product Size (bp)

Allelic Ladder

Sample

#2

Sample

#1

TCCCAAGCTCTTCCTCTTCCCTAGATCAATACAGACAGA

AGACAGGTGGATAGATAGATAGATAGATAGATAGATAGA

TAGATAGATAGATATCATTGAAAGACAAAACAGAGATGG

ATGATAGATACATGCTTACAGATGCACAC

PCR primers anneal to unique sequences

bracketing the variable STR repeat region

= 11 GATA repeats (“11” is all that is reported)

The overall PCR product size is measured

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

13 Core U.S. STR Loci

AMEL

AMEL

Sex-typing

Position of Forensic STR Markers

on Human Chromosomes

8 STR loci overlap between U.S. and Europe

1997

Family Inheritance of STR Alleles (D13S317)

Father

Child #1

Child #2

Child #3

Mother

PCR product size (bp)

11 14

11

12 14

8 14

12

12 8

PATERNITY TESTING

Why we use PCR?

+/- How

much?

(Qualitative)

(Quantitative)

A restriction enzymes binds to DNA at a specific sequence and make a double-

stranded cut at or near that sequence.

Restriction endonucleases cut DNA

molecules at defined positions



• Sequencing

• RFLP analysis

• Probes

– Southern blot

– Dot blot

– Microarray

•PCR

•Gel

electrophoresis

RFLP Analysis

RFLP (Restriction Fragment Length Polymorphism) analysis relies on

the use of restriction enzymes

These enzymes recognize specific DNA sequences (usually

palindromes) and cut them…

RFLP Analysis Restriction Fragment Length Polymorphism means that there are

polymorphisms (differences) between people in the number of restriction

sites (and therefore the length of the cut fragments). This person has 4

fragments after restriction digest.

This person has a mutation that eliminates one of the sites that the restriction

enzyme cuts at. Therefore, this person has 3 bands, one of them being much

larger than the rest. If this mutation was associated with a disease, a restriction

digest would show that a carrier of the mutation had 3 fragments instead of 4

RFLP Analysis

RFLP Analysis

Genomic DNA is very long and

contains a lot of restriction

enzyme cut sites

PCR makes many

copies of a small

region of DNA,

better for RFLP

analysis

RFLP Analysis

Issues with RFLP analysis

• Can not detect all mutations – the

mutation has to coincide with a

restriction enzyme cut site

F

V

LUUR

T

P

E

LCUN

SAGY

H

ND5ND6

ND4

ND4L

ND3R

COIII

G

KCOIID

SUCN

COI

AN

CY

W

ND2

I

M

Q

ND1

16S

12S

D-loopCyt.b

3460G-A

11778G->A

14484T->C

LHON

DDEE

LL

EE

TT

II

OO

NN

3243A->G

MELAS

CPEO

DDM

8344A->G

MERRF

tRNA-Ser mutations

DEAFNESS

cyt. b mutations

MYOGLOBINURIC

MYOPATHY

8993T->G

NARP/MILS

PEO, KSS, Pearson

1555A->G

DEAFNESS (aminoglycosides)

tRNA-Ile mutations

CARDIOPATHY

ATPase6/8

F

V

LUUR

T

P

E

LCUN

SAGY

H

ND5ND6

ND4

ND4L

ND3R

COIII

G

KCOIID

SUCN

COI

AN

CY

W

ND2

I

M

Q

ND1

16S

12S

D-loopCyt.b

3460G-A

11778G->A

14484T->C

LHON

DDEE

LL

EE

TT

II

OO

NN

3243A->G

MELAS

CPEO

DDM

8344A->G

MERRF

8344A->G

MERRF

tRNA-Ser mutations

DEAFNESS

cyt. b mutations

MYOGLOBINURIC

MYOPATHY

8993T->G

NARP/MILS

8993T->G

NARP/MILS

PEO, KSS, Pearson

1555A->G

DEAFNESS (aminoglycosides)

tRNA-Ile mutations

CARDIOPATHY

ATPase6/8

mtDNA Disorders

Introduction

The mobility in gel electrophoresis of double-stranded DNA's of a given

length is relatively independent of nucleotide sequence.

the mobility of single strands can

vary considerably as a result of only

small changes in nucleotide sequence.

SSCP techniques may be used as

Screening methods to point to the mutations

Most SSCP protocols are

designed to analyze the polymorphism at single loci. To

this end a specific pair of PCR

primers bracketing the target

region is used to amplify DNA from individuals

Gel Discussion • Number of bands in SSCP: 2 – 8

and even more.

• Reason: This maybe due to

different shapes and conformations

of single strands.

http://www.clinchem.org/cgi/content/full/43/1/30/F1

For avoiding of returning single strands to

double form, strands should be pushed

into gel rapidly .For this, initial 15 minutes

should have 350 voltage and then return it

to 60-120.

There is two methods for detecting a

mutation into a sample:

SSCP Up to 80% Sequencing Up to 99%

Principle of DHPLC

Rapid denaturation of DNA by heating, re-annealing by slow cooling.

Heteroduplexe

s form in the presence of two different alleles.

Homoduplexes

Heteroduplexes

Chromatographic Separation

Reverse-phase ion-pair system. Gradient elution with acetonitrile/water. UV detection.

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution.

Reverse transcriptase uses a single-stranded RNA template to create a

double-stranded DNA.

What does reverse transcriptase do?

copy

c c c

//upload.wikimedia.org/wikipedia/en/8/80/One-step_vs_two-step_RT-PCR.jpg

Let’s start!

total RNA

tRNA rRNA mRNA

~ 1%

• Most of the RNA is unimportant for us (tRNA, rRNA)

• mRNA population consists of about 3-5000 different kind

• Strong secondary structure – enzyme cannot work

AAAAA

Only mRNA has a poly-Adenin tail at the 3’ end

RNA isolation

RT reaction – step by step

AAAAA

AAAAA

AAAAA

65 ºC + ice

37 ºC

TTTTT

TTTTT

TTTTT

TTTTT

Reverse

transcriptase

dATP

dCTP

dGTP

dTTP

RNase

inhibitor

mRNA

mRNA

1. denature

AAAAA TTTTT mRNA

2. anneal +

elongate

TTTTT cDNA AAAAA

37 ºC

RT

cDNA

RT

RT

ready

How to amplify our gene of interest from the cDNA “soup”?

TTTTT cDNA AAAAA

TTTTT AAAAA cDNA TTTTT

AAAAA cDNA

PCR

Gel

visualization

Gene

#2

750 bp

500 bp

Gene

#1

Gene-specific

primers

RT–PCR at the bench

total RNA +

oligodT

37 ºC – 1 hour

anneal +

elongate

65ºC – 10 min

denature

Add:

Enzyme

dNTPs

RNasin RT

ready

RT:

PCR:

DNA pol

dNTPs

primers

Buffer

MgCl2

95ºC

3 min

denature amplify

95ºC – 30 sec

55ºC – 30 sec

72ºC – 1 min

72ºC

10 min

finish

PCR

ready

template

Gel analysis

30 cycles

Sanger Sequencing

DNA Sequencing: Dideoxy

Method

• Modified sugars cause chain

termination because it lacks the 3’-OH

group, which is essential for

attachment of the next nucleotide in a

growing DNA strand

• The products of DNA synthesis are

then separated by electrophoresis. In

principle, the sequence can be read

directly from the gel

Analysis of Recombinant Clones: Thermal Cycle DNA Sequencing

O

H H

H OH

H

Base

H

PO4

O

H H

H H

H

Base

H

PO4

dNTP

ddNTP



• Sequencing

• RFLP analysis

• Probes

– Southern blot

– Dot blot

– Microarray

•PCR

•Gel

electrophoresis

Sequencing

aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg

“Known gene sequence:”

*******************************************

Patient DNA sequence

How do we find out if the patient’s

sequence is different?

Sequencing

*******************************************

tttttttt actactactac*

tttttttt

tttttttt

tttttttt

*******************************************

*******************************************

*******************************************

******************************************* tttttttt

actac*

actactactactag*

actactactactacgactact*

actactactacta*

Sequencing

*******************************************

tttttttt a*

*******************************************

*******************************************

*******************************************

*******************************************

tttttttt actac*

tttttttt act*

tttttttt ac*

tttttttt acta*

Sequencing

*******************************************

tttttttt a*

*******************************************

*******************************************

*******************************************

*******************************************

tttttttt actac*

tttttttt act*

tttttttt ac*

tttttttt acta*

a

Sequencing

*******************************************

tttttttt a*

*******************************************

*******************************************

*******************************************

*******************************************

tttttttt actac*

tttttttt act*

tttttttt ac*

tttttttt acta*

a c

Sequencing

*******************************************

tttttttt a*

*******************************************

*******************************************

*******************************************

*******************************************

tttttttt actac*

tttttttt act*

tttttttt ac*

tttttttt acta*

t a c

Sequencing

*******************************************

tttttttt a*

*******************************************

*******************************************

*******************************************

*******************************************

tttttttt actac*

tttttttt act*

tttttttt ac*

tttttttt acta*

t a c a

Sequencing

*******************************************

tttttttt a*

*******************************************

*******************************************

*******************************************

*******************************************

tttttttt actac*

tttttttt act*

tttttttt ac*

tttttttt acta*

t a c a c…

Sequencing

actactactactagtactactactactactactac

*******************************************



Sequencing

actactactactagtactactactactactactac aaaaaaaatgatgatgatgatcatgatgatgatgatgatgatg



Mutation

Issues with Sequencing

• Informative

• Time consuming

• Expensive

Electrophoresis

Southern Blot

Types of Blots

• Southern Blot – use DNA to probe DNA

• Northern Blot – use DNA to probe RNA

• Western Blot – use antibodies to probe

Protein

The Southern blotting technique

Southern Blot

Restriction enzyme

DNA of various sizes

Electrophorese on agarose gel

gel

Denature - transfer to filter paper.

blot

Hybridize to probe

Visualize

Denature- transfer to filter paper.

blot

CGG REPEATS IN FRAGILE X SYNDROME

NORMAL 5-50

PREMUTATION 60-200

AFFECTED >200

(CGG)N

FMR-1 GENE

Structure and inheritance of CTG repeats

in myotonic dystrophy

normal 5-30

premutation 45-75

affected >75

(CTG)n

myotonic protein kinase gene

MLPA

• Detection of aberrant copy number of 45 genomic DNA sequences in one easy to perform, PCR based reaction.

• Minimum of only 20 ng DNA

• Partially degraded DNA

– DNA extracted from paraffin

– Formalin treated tissues

– Free fetal DNA obtained from maternal plasma

• Discriminates sequences that differ in only a single nucleotide.

• 45 different mRNAs

• To determine the methylation status of promoters

• Detection of known mutations and SNPs.

MLPA technique

1. Denaturation

2. Hybridization

3. Ligation

4. Amplification

SALSA MLPA probes

Hybridysation

1. The MLPA probemix is added to denatured genomic DNA

2. The two parts of each probe hybridise to adjacent target sequences

Ligation

3. Probes are ligated by a thermostable ligase

Amplification

4. A universal primer pair is used to amplify all ligated probes.

The amplification product of each probe has a unique length (130 480 bp).

Separation and quantification by capillary

electrophoresis

Each peak is the

amplification product

of a specific probe.

Samples are

compared to a control

sample.

A difference in

relative peak height

or peak area

indicates a copy

number change of the

probe target

sequence

Triple X

Female

Male

283 bp 346 bp

X

control

Homozygous deletion ASPA exon 1-6

1 5 4 3 2 6

1

5

4

3

2 6

Specificity

of MLPA

probes is

very high.

Heterozygous ASPA del. exon 1-6

Control

1

5

4

3

2 6

1 5 4 3 2 6

Reproducibilit

y of MLPA is

sufficient to

distinguish

homozygotes

and

heterozygotes.

55 oC hybridisation

60 oC hybridisation

62 oC hybridisation

Many variables have only a small influence on MLPA results

10 ng. DNA

(less than recommended minimum amount)

50 ng. DNA

750 ng. DNA

Amount of DNA used has little

influence on MLPA results

Detection of point mutations

• Only perfectly matched probes will

be ligated Probes for common

mutations or SNP’s can be made

–Signal will only be present when the

mutation is present

Normal

Ligation of the two probe oligonucleotides

Amplification product

Mismatch at the probe ligation site

No ligation, no amplification product

MLPA discriminates sequences that differ

in only a single nucleotide and can be used

to detect known mutations.

Mismatch Perfect match

Advantages of MLPA

• Detection of copy number of 45 genomic DNA sequences in a simple to perform, PCR based, reaction.

• Requires only 20 ng human DNA

• Discriminates sequences that differ in only a single nucleotide.

• Only a thermocycler and a sequence type electrophoresis system are required.

• Identical protocol for many different applications.

• High throughput ; Results available within 24 hrs.

• Large lot sizes (> 100.000 reactions) are possible. All reagents have proved to be very stable.

• For each probe signal, presence of two specific oligonucleotides is required.

• All reagents are fluid: Simplified quality control.

• Including electrophoresis, total reagent costs are < EUR 15,- / reaction.

Disadvantages of MLPA

• Results depend on sample quality. For amniotic fluid samples we recommend to use cell lysates rather than purified DNA.

• Amniotic fluid samples that are contaminated with maternal blood can not be used.

• MLPA can not detect all triploidies.

• MLPA can not detect balanced translocations. More than 130 MLPA tests are currently available from MRC-Holland.

• MRC-Holland is research oriented and is not yet ISO certified.

• MLPA kits are not yet CE certified.

• Time consuming and difficult to develop new MLPA based assays.

MLPA products

• Detection of aneuploidy of chromosomes 13, 18, 21, X and Y

• Detection of large chromosomal deletions or duplications: DiGeorge syndrome, Williams syndrome, Spinal Muscular Atrophy (SMA), subtelomeric regions etc

• Detection of gains and losses of genes in cancer tissues: Her2-neu (ERBB2), TP53, MYC etc.

• Sequencing

• RFLP analysis

• Probes

– Dot blot

– Microarray

•PCR

•Gel

electrophoresis

Probes

DNA bases pair with complementary bases

Probes

Probe

A probe is an a short fragment of DNA that is complementary to part

of a longer DNA sequence.

DNA strands can be

separated with high

heat.

As the temperature is

lowered, smaller

fragments that have

complementary

sequences (probes) will

base pair faster than the

long original strands of

DNA

Probes The ability of a probe to bind depends on:

*Its complementarity to the DNA strand it’s binding to…

-Single base pair differences can affect binding

depending on…

*The stringency of the binding conditions

-Temperature

-Buffer conditions

Low stringency

High stringency

• Sequencing

• RFLP analysis

• Probes

– Dot blot

– Microarray

•PCR

•Gel

electrophoresis

Dot Blot (A Southern blot without the gel…)

A patient’s DNA (genomic DNA or

PCR products) is denatured and

spotted to a membrane…

Dot Blot

Membrane is washed in a

solution that contains

radioactively labeled probe

Probe binds to

complementary

sequence

Dot Blot

Membrane is washed in a

solution that contains

radioactively labeled probe

Excess probe is washed off

– only probe bound to DNA

on the membrane remains

Probe binds to

complementary

sequence

Dot Blot

The membrane is exposed to

autoradiography film.. Therefore,

wherever there is radioactive probe, the

film will be exposed…

Dot Blot

The membrane is exposed to

autoradiography film..

Therefore, wherever there is

radioactive probe, the film will

be exposed…

And a black dot will be

developed where the probe

was…

Dot blot to diagnose cystic fibrosis (CF)

Normal

Normal

probe

Mutant

probe

CF patient

(homozygous mutant)

CF carrier

(heterozygous mutant)

DNA from a patient is spotted out twice, one will be used with a

probe complementary to the normal sequence, the other will be used

with a probe that is complementary to a mutated sequence.

Since a heterozygote has

one copy of the normal

gene and one copy of the

abnormal gene, both

probes can bind, but only

half as much binds,

making the dot lighter.

Revers

e D

ot B

lot

Issues with dot blots

• Can be used to test patient for

multiple mutations

• Single base pair mutations may be

hard to detect with dot blots

Functional genomics = The ability to perform

genome-wide patterns of gene expression

and the mechanisms by which gene

expression is coordinated.

DNA microarrays can be used to detect

differences in the levels gene expression in

different populations of cells on a genome-

wide level.

Reverse Transcriptase

in vitro transcription

mRNA

cDNA

Fragmentation of

cRNA

cRNA

GeneChip

Hybridization

This is what

we are doing

in this class

Hybridization and Staining

Array

Fragmented cRNA Target

RNA:DNA Hybridized Array

Streptavidin phycoerythrin

[Fluorescent dye]



• Sequencing

• RFLP analysis

• Probes

– Dot blot

– Microarray

•PCR

•Gel

electrophoresis

Microarray (High throughput dot blots. Allows for testing of many different mutations…)

Microarrays start

with a “chip” on

which is spotted

many different

probes for different

mutations. Each

dot represents a

different probe.

If this were a dot

blot, it would be

called a “reverse

dot blot” because

the PROBE is

spotted, rather than

the DNA.

Microarray (High throughput dot blots. Allows for testing of many different mutations…)

The chip is treated

much like a southern

or a dot blot, except it

is washed with a

patient’s DNA

(fragmented with

restriction enzymes,

denatured and

labeled with a

flourescent dye), and

it automated.

Microarray

Binds to a probe for a

CF mutation


colon-cancer

susceptibility mutation


breast-cancer

susceptibility mutation

If a patient’s DNA binds to spotted probes, a computer detects this by the

measuring the intensity of the flourescence emanating from that spot. A

homozygote for a CF mutation will have a more intense spot than a

heterozygote for the same mutation

Issues with Microarrays

• Expensive

Karyotyping

FISH

Biomedical Genetics

Molecular Genetics

1400 nm

700 nm

300 nm

30 nm

11 nm

2 nm

Dr. M. Houshmand

PGD

Achondroplasia

Adrenoleukodystrophy

Agammaglobulinemia

Alpha-1-Antitrypsin

Alpha Thalassemia

Alport Disease

Alzheimer's disease - Early onset (PSEN1-2)

Becker muscular dystrophy

Beta Thalassemia

Charcot Marie Tooth

Chromosomal aneuploidies by FISH

Cystic Fibrosis

Cruzon syndrome

Duchenne muscular dystrophy

Dystonia

Epidermolysis Bullosa

Fanconi Anemia

Familial adenomatous polyposis (FAP)

Familial dysautonomia

Fragile-X syndrome

Gaucher’s Disease

Glycogen storage disease

Hemophilia A and B

HLA typing

HSNF5 mutations

Huntington disease

Hurler syndrome

Incontinentia pigmentii

Kell disease

Marfan syndrome

MELAS

Multiple Endocrine Neoplasia Type II (MEN II)

Multiple Epiphysial Dysplasia

Myotonic Dystrophy

Myotubular myopathy

Neurofibromatosis type I

Neurofibromatosis type II

Norrie disease

Osteogenesis imperfecta I - IV

OTC Deficiency

P53 Oncogene

Phenylketonuria

Polycystic kidney disease (Autosomal Dominant

types I and II)

Retinitis Pigmentosa

SCA 6

Sickle Cell Anemia

Sonic hedgehog mutations

Spinal/Bulbar Muscular Atrophy

Spinal Muscular Atrophy

Tay-Sachs Disease

Translocations by FISH

Tuberous sclerosis

Von Hippel Lindau

Wiskott-Aldrich syndrome

X linked Disease by sexing

X-linked hydrocephalus

X-linked hyper IgM syndrome

Current Techniques Applied to

Molecular Pathology

(one gene – one disease)

• Southern blot

• Dot blot/Reverse dot blot

• Polymerase chain reaction

• SSCP/DGGE

• RT-PCR

• DNA sequencing

• TaqMan, real-time PCR

• Invader assay

• In situ hybridization

New Techniques Coming to

Molecular Pathology

(all genes – all diseases)

• Southern blot

• Dot blot/Reverse dot blot

• Polymerase chain reaction

• SSCP/DGGE

• RT-PCR

• DNA sequencing

• TaqMan, real-time PCR

• Invader assay

• In situ hybridization

• Microarray hybridization

• High-density microarray hybridization

• Array comparative genomic hybridization

• Whole-genome sequencing

Difficulties:

Allelic drop out

Contamination

Background for each

Disease

Materials and

Equipments

Information

Dr. M. Houshmand

Refferal Lab

User Lab 5

Service Lab 1

User Lab 4

User Lab 1

User Lab 3

User Lab 2 Service Lab 2

Service Lab 3

Service Lab 4

Service Lab 5

genetic diagnostic method - انجمن علمی دکترای علوم ... to generate a case report...

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