[CYTOGENETICS] FLUORESCENCE IN-SITU HYBRIDIZATION (FISH)

FLUORESCENCE IN-SITU HYBRIDIZATION

(FISH)

Same as karyotyping (purpose)

Specializes in specificity

FLUORESCENCE IN-SITU HYBRIDIZATION

(FISH)

Can be used to detect what specific parts are deleted or duplicated

Study certain sequences

True

T/F:

In karyotyping, we cannot locate sequences (lacks specificity)

Hybridization

In FISH, we first denature the DNA molecule before we can study the interest

We are to facilitate the formation of new bonds

FLUORESCENCE IN-SITU HYBRIDIZATION

(FISH)

a cytogenetic technique that uses fluorescent probes that bind specifically to a part of chromosomes complementary to their sequence

Useful in detecting and mapping the presence or absence of particular DNA sequences within chromosomes

FLUORESCENCE IN-SITU HYBRIDIZATION

(FISH)

applied to provide the specific localization of genes on chromosomes

Rapid diagnosis of trisomies and microdeletions is acquired using specific probes

FLUORESCENCE IN-SITU HYBRIDIZATION

(FISH)

Enable us to detect trisomies and microdeletions

True

T/F;

in FISH, We are studying the chromosome (intact)

Interphase and Metaphase

Two phases of cell division where we can do FISH

Know the location of the DNA you are specifically interested in

First step of FISH

NCBI

lists all the locations of the DNA

100-1,000 nucleotides

number of fluorescent labeled probes to develop

The longer the probe, the greater the specificity; Better visualization

Heat the cells at 90-95 degree Celsius (to destroy Hydrogen Bonds)

How do we denature and separate the DNA strands and allow probe access to the target DNA

Probes can bind to them when denatured

Unique from the karyotype

Cool down to 65-70 degree celsius the probe

How do we hybridize or facilitate new bonds

Purpose: make new bonds

But the DNA can also renature on itself (possible)

Lower or cooler temp

Longer probes to hybridize require what temperature

Fluorescent microscope

Microscope used in FISH

1) Locate the DNA

2) Generate complementary sequence

3) Denature the DNA

4) Hybridization

5) Analyze

Summary of steps of FISH

UV light

The fluorescent tag needs to be stimulated by _____ light because it is not visible to the naked eye (for fluorescent)

Somatic cells

Cells used in FISH (Somatic or Sex)

Metaphase Cells

Gold standard and routinely done

Done on cultured cells

Allows direct visualization of chromosomes and exact position of signals

> Can detect translocations

Metaphase Cells

Useful in the detection of structural changes in the genome

> Arranged from biggest to smallest

Amniocytes

Chorionic villous cells

Lymphocytes

Cells from bone marrow aspirates or solid tumors

Fibroblasts

Samples of metaphase cells

Interphase Cells

May also be done on uncultured specimens

Advantageous in the rapid screening of many nuclei for prenatal diagnosis and newborn studies (fast TAT)

> We do not have to culture the cells anymore

> Mas madali by 48-72hours

48-72hours

Hours we can save if we use interphase cells in FISH

Interphase Cells

Also beneficial in the study of samples with a low mitotic index, such as most solid tumors

Interphase Cells

Major disadvantage is the inability to detect unknown structural chromosomal changes

You can never find out translocation

Amniocytes

for ploidy analysis during prenatal studies

Peripheral blood smears

for ploidy analysis in newborns

Bone marrow aspirate smear or direct harvest

translocation, or copy number analysis in cancer studies

Duplication

We we see 2 nuclei in the interphase, we can deduce a _______ type of aberration

Deletion

What can be the interpretation if we cannot see any nucleus in the interphase cells of FISH

Urinary and Respiratory tract

Amniocytes can be harvested from the epithelial cells of what tract or tissues of fetus

Cells from bone marrow aspirates or solid tumors

Rare if we are dealing for some types of lukemia

We see more immature cells rather than the peripheral blood (matured cells)

FISH probes

Complementary sequences of target nucleic acids (DNA, RNA, or nucleic acid analogs) tagged or labeled with fluorophores

Size ranges from 20 to 1000 base pairs (20-25 starting will be OK for PCR)

Direct Labeling

fluorophores are directly attached to the probe

less sensitive but easier to perform

Ex. FITC, rhodamine, and cyanines

Indirect Labelling

chemical conjugation of the nucleic acid with a nonfluorescent molecule that can bind fluorescent material after hybridization

They are like molecular bridges

Needs a binding molecule in a form of biotin

Ex. Biotin and Digoxigenin

5' end of the DNA

What end of the DNA ang nilalagyan ng probes

True

T/F:

Not every nucleotide is fluorescent; Just enough to produce visible light

90-95 degree celsius

How hot is the temp for denaturing DNA

Locus Specific Fish Probes

Used if we want to know deletions and translocations

Metaphase probe

Binds to a particular region of a chromosome

Used when only a small portion of a gene is isolated and wants to determine on which chromosome the gene is located, or how many copies of a gene exist within a particular genome

Single color FISH probe

Designed to cover a gene of interest

Dual color FISH probe

Designed to cover any 2 genes for the detection of any aberrations.

Allows simultaneous detection of numerical abnormalities of two to three regions in one FISH assay

APHLOID OR CENTROMERIC REPEAT

For deletion

Parang C-banding

Generated from repetitive sequences found in the middle of each chromosome

Used to determine whether an individual has the correct number of chromosomes or if there is aneuploidy in the patient’s genome

APHLOID OR CENTROMERIC REPEAT

Example application:

Magagamit to check for abnormality by counting the centromere.. For example, ang human ay may 23 pairs, so dapat may 46 centromeres lang ang makikita mo. Pag 48 nakita mo then may idea ka na either may aneuploidies or dicentric chromosome.

SUBTELOMERIC PROBE

Locate the subtelomere

Specific to the subtelomere region of chromosome

Useful in the detection of subtelomere deletions and rearrangements

Whole Chromosome Probe

A collection of smaller probes that bind to the whole length of the chromosome

Useful in the examination of chromosomal aberrations

(Sir clive thinks that this is used if the DNA is in between the p-arm and the q-arm and he does not see the purpose of using whole chrome probes)

PRE-NATAL FISH PROBES

Stargets chromosome 13,18,21 X, and Y

Because this is prone to trisomies of duplication and any aberrations

formamide/ salt solution

FIRST STEP

Probe and target DNAs are denatured using high-temperature incubation in a ________

stringent washing

SECOND STEP

Probe sequences hybridize to the complementary target sequences, and nonspecific binding is eliminated via ____________

fluorescence microscopy

THIRD STEP

The probe hybridization is detected with ______________ microscopy

Not diluted properly with hypotonic saline solution

Reason why FISH specimen are overlapping

Duplication

In the event that only green is seen (color), it is interpreted as a ________ type of aberration.

Deletion

In the event that only red is seen (color), it means there is a ________.

Yellow Color

Color of normal FISH

3'-5'

Direction of STRAND OF DNA IN A MICRO-ARRAY

5-3

Direction of

GREEN: ORIGINAL STRAND

RED: COMPLIMENTARY STRAND

COMPARATIVE GENOMIC HYBRIDIZATION (CGH) on METAPHASE CELLS

We have to extract DNA

Good for deletions and duplication, only (gain or losses)

COMPARATIVE GENOMIC HYBRIDIZATION (CGH) on METAPHASE CELLS

a technique that uses DNA from the cells of interest, rather than using a standard karyotype, for chromosomal analysis.

This can be very useful, especially in some cancers when only DNA is available rather than any growing cells.

COMPARATIVE GENOMIC HYBRIDIZATION (CGH) on METAPHASE CELLS

This technology has been used successfully for clinical analysis, particularly with cases that have a low (or no) mitotic index.

It is not useful for detecting balanced rearrangements

Multiplex FISH (M-FISH)

A technique that allows the investigator to view a karyotype so that each chromosome is “painted” with a different color

Ratio-labeled probes are used to create a distinct computer-generated false color for each chromosome

Useful for complex rearrangements, such as those seen in neoplastic disorders and solid tumors

Multiplex FISH (M-FISH)

If you observe the image, particularly chromosome 4, makikita mong may translocation. Dun sa may bandang dulo nya, nag-iba ‘yung color. ‘Yung part na ‘yon (red) ay galing sa chromosome 11. Nagkaroon ng translocation ‘yung chromosome 11 papunta sa 4.

Multicolor Banding Analysis

Uses chromosome-specific mixtures of partial chromosome paints that are labeled with various fluorochromes

A computer program analyzes metaphase chromosome data and produces a pseudocolored, banded karyotype with an estimated resolution of 550 bands, regardless of chromosome length

Multicolor Banding Analysis

Advantageous for the determination of breakpoints and the analysis of intrachromosomal rearrangements, and can be particularly useful in preparations with shorter chromosomes

image B (in the q arm - no corresponding colors)

between A and B which is abnormal as per multicolor banding analysis?

Fiber FISH

A technique that is almost entirely used for research

It allows the chromosomes to be stretched out and elongated

The probes are applied and can be physically ordered on the fibers

This provides a much higher spatial resolution and allows for correct orientation and placement of probes and for precise mapping of probes

True

T/F:

for Fiber FISH analysis, the stretched out part or highlighted part is usually the telomere

Primed In Situ Labeling (PRINS)

Essentially, PCR on a slide

Primers of interest are hybridized on a slide and then subjected to cycles of denaturation, reannealing, and elongation that are used to incorporate labeled nucleotides. The labels are then detected fluorescently, or labeled nucleotides are incorporated during the reaction

Can differentiate hybridization with the alpha satellite sequences for chromosomes 13 and 21, something that cannot be done with traditional FISH

Reverse FISH

Used to identify material of unknown origin

This unidentified material, such as a marker chromosome or duplication, is flow sorted or microdissected off a slide after G-banding. The DNA from this material is extracted, PCR-amplified, and labeled with a fluorochrome. This is then used as a probe and hybridized to normal or patient metaphase chromosomes to identify the origin of the unknown material

Reverse FISH

Patient with abnormal chromosome 8 is targeted.

Ginawa muna 'yung typical karyotyping.

● Additional bands are microdissected and amplify the particular part lang!

● Parang nag-reverse banding ka muna then nadetect mo sya by banding techniques, then 'yung additional band, you use that to prepare the probe for your FISH

● BANDING TECHNIQUE NG KARYOTYPE FOLLOWED BY FISH