Τεχνικές της Μοριακής Βιολογίας

Τεχνικές της Μοριακής Βιολογίας
Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών Σχολή Θετικών Επιστημών Τμήμα Βιολογίας Τομέας Βιοχημείας και Μοριακής Βιολογίας Τεχνικές της Μοριακής Βιολογίας Μέρος 3ο Χρήστος Κ. Κοντός, Ph.D. Επίκουρος Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.

in situ υβριδισμός (ISH)
in situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA (cDNA), RNA (cRNA) or modified nucleic acids (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry (IHC), which usually localizes proteins in tissue sections. in situ hybridization is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding the organization, regulation, and function of genes.

The key techniques currently in use include: in situ hybridization to mRNA with oligonucleotide and RNA probes analysis with light and electron microscopes whole mount in situ hybridization double detection of RNAs and RNA plus protein fluorescent in situ hybridization to detect chromosomal sequences DNA ISH can be used to determine the structure of chromosomes. Fluorescent DNA ISH (FISH) can, for example, be used in medical diagnostics to assess chromosomal integrity. RNA ISH (RNA in situ hybridization) is used to measure and localize RNAs (mRNAs, lncRNAs, and miRNAs) within tissue sections, cells, whole mounts, and circulating tumor cells (CTCs).

in situ υβριδισμός RNA KRT5 και γονίδιο housekeeping σε ιστολογική τομή FFPE από ανθρώπινο μελάνωμα [οπτικό πεδίο από οπτικό μικροσκόπιο (αριστερά) και μικροσκόπιο φθορισμού (δεξιά)].

in situ υβριδισμός RNA του γονιδίου hb (hunchback) σε διάφορα αναπτυξιακά στάδια εμβρύων Drosophila αγρίου τύπου.

Nick translation Nick translation is a tagging technique.
DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence. Λειτουργικές δομικές περιοχές (domains) στο θραύσμα Klenow (αριστερά) και τη DNA πολυμεράση I (δεξιά).

Nick translation Nick translation is a tagging technique.
DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used: as a probe in fluorescent in situ hybridization (FISH), as a probe in blotting techniques, for radiolabeling. To radioactively label a DNA fragment for use as a probe in blotting procedures, one of the incorporated nucleotides provided in the reaction is radiolabeled in the α-phosphate position, e.g. ATP, [α-32P]. Similarly, a fluorophore can be attached instead for fluorescent labelling, or an antigen for immunodetection.

Nick translation

Nick translation H nick translation μπορεί να χρησιμοποιηθεί για τη σήμανση (labeling) ενός μορίου DNA με φθορίζουσα ουσία ή ραδιενέργεια.

in vitro transcription
A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.

Φθορίζων in situ υβριδισμός (FISH)
Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s and is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

DNase Εφαρμογή του φθορίζοντος in situ υβριδισμού (fluorescent in situ hybridization, FISH) για τον εντοπισμό ενός γονιδίου στον πυρήνα.

Ανίχνευση πολλαπλών RNA σε κύτταρα με χρήση της μεθόδου FISH.

Ανίχνευση του miR-133 (πράσινο) και του mRNA της μυογενίνης (κόκκινο) σε κύτταρα C2C12 που διαφοροποιούνται.

Ουροθηλιακά κύτταρα, σεσημασμένα με 4 διαφορετικούς δείκτες (markers).

Έλλειψη του γονιδίου TP53. Τρισωμία 21 (αιτία του συνδρόμου Down). Έλλειψη μιας περιοχής στο χρωμόσωμα 17 (αιτία του συνδρόμου Smith-Magenis). Μεταφασικά χρωμοσώματα, όπου διακρίνεται η σύντηξη BCR/ABL1 (χρόνια μυελογενής λευχαιμία).

Χρωμογόνος in situ υβριδισμός (CISH)
Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER2 oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH.

Χρωμογόνος in situ υβριδισμός (CISH)
Διαδικασία για τη διεξαγωγή χρωμογόνου in situ υβριδισμού (Chromogenic in situ hybridization, CISH).

vs. Χρωμογόνος in situ υβριδισμός (CISH) Σύγκριση συστημάτων ανίχνευσης που χρησιμοποιούνται σε FISH (άμεση ή έμμεση ανίχνευση) και CISH (έμμεση ανίχνευση).

vs. Χρωμογόνος in situ υβριδισμός (CISH) in situ υβριδισμός RNA KRT5 και γονίδιο housekeeping σε ιστολογική τομή FFPE από ανθρώπινο μελάνωμα [οπτικό πεδίο σε μικροσκόπιο μετά από CISH (αριστερά) και μετά από FISH (δεξιά)].

Συγκριτικός γονιδιωματικός υβριδισμός (CGH)
Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analyzing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions (a portion of a whole chromosome). This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH), which are limited by the resolution of the microscope utilized.

Διαδικασία συμβατικού συγκριτικού γονιδιωματικού υβριδισμού (comparative genomic hybridization, CGH)

Through the use of DNA microarrays in conjunction with CGH techniques, the more specific form of array CGH (a-CGH) has been developed, allowing for a locus-by- locus measure of CNV with increased resolution as low as 100 kilobases. This improved technique allows for the etiology of known and unknown conditions to be discovered. Array CGH has proven to be a specific, sensitive, fast and high-throughput technique, with considerable advantages compared to other methods used for the analysis of DNA copy number changes making it more amenable to diagnostic applications. Using this method, copy number changes at a level of 5–10 kilobases of DNA sequences can be detected. As of 2006, even high-resolution CGH (HR-CGH) arrays are accurate to detect structural variations at resolution of 200 bp. This method allows one to identify new recurrent chromosome changes such as microdeletions and duplications in human conditions such as cancer and birth defects due to chromosome aberrations.

Διαδικασία συγκριτικού γονιδιωματικού υβριδισμού σε συστοιχία [array comparative genomic hybridization, array CGH (a-CGH)]

Συμβατικός καρυότυπος Καρυότυπος άνδρα με χρώση Giemsa.

Εικονικός καρυότυπος (virtual karyotype)
Virtual karyotype is the digital information reflecting a karyotype, resulting from the analysis of short sequences of DNA from specific loci all over the genome, which are isolated and enumerated. It detects genomic copy number variations at a higher resolution level than conventional karyotyping or chromosome-based comparative genomic hybridization (CGH). The main methods used for creating virtual karyotypes are array- comparative genomic hybridization (a-CGH) and SNP arrays.

Εικονικός καρυότυπος (virtual karyotype) ενός δείγματος ασθενούς με χρόνια λεμφοκυτταρική λευχαιμία, με χρήση συστοιχίας SNP (SNP array).

Διάγραμμα αναλογίας log2 (log2 ratio) εικονικού καρυότυπου ενός δείγματος ασθενούς με χρόνια λεμφοκυτταρική λευχαιμία, με χρήση συστοιχίας SNP (SNP array). Αριθμός αντιγράφων: Κίτρινο = 2 (φυσιολογικός/διπλοειδής) Γαλάζιο = 1 (έλλειψη) Ροζ = 3 (τρισωμία).

Διάγραμμα αναλογίας log2 (log2 ratio) εικονικού καρυότυπου ανθρώπινου δείγματος, όπου διακρίνεται ο αυξημένος αριθμού αντιγράφων του γονιδίου HER2 και η έλλειψη του γονιδίου WNT3.

Spectral Karyotyping (SKY)
What is SKY? Spectral karyotyping (SKY) is a laboratory technique that allows scientists to visualize all of the human chromosomes at one time by "painting" each pair of chromosomes in a different fluorescent color.

How does SKY work? SKY involves the preparation of a large collection of short sequences of single- stranded DNA called probes. Each of the individual probes in this DNA "library" is complementary to a unique region of one chromosome - together, all of the probes make up a collection of DNA that is complementary to all of the chromosomes within the human genome. Each probe is labeled with a fluorescent color that is designated for a specific chromosome. For example, probes that are complementary to chromosome 1 are labeled with yellow molecules, while those that are complementary to chromosome 2 are labeled with red molecules, and so on. When these probes are mixed with the chromosomes from a human cell, the probes hybridize to the DNA in the chromosomes. As they hybridize, the fluorescent probes essentially paint the full set of chromosomes in a rainbow of colors. Scientists can then use computers to analyze the painted chromosomes to determine whether any of them exhibits translocations or other structural abnormalities.

What is SKY used for? Many diseases are associated with particular chromosomal abnormalities. For example, chromosomes in cancer cells frequently exhibit aberrations called translocations, in which a piece of one chromosome breaks off and attaches to the end of another chromosome. Identifying such chromosomal abnormalities and determining their role in disease is an important step in developing new methods for diagnosing many genetic disorders. Traditional karyotyping allows scientists to view the full set of human chromosomes in black and white, for observing the number, size and shape of the chromosomes. Interpreting these karyotypes, however, requires an expert, who might need hours to examine a single chromosome. By using SKY, even non-experts can easily see instances where a chromosome, painted in one color, has a small piece of a different chromosome, painted in another color, attached to it.

What it detects: Microscopic (>5-10 Mb) genomic abnormalities Balanced and unbalanced translocations Aneuploidies What it doesn’t detect: Submicroscopic abnormalities (<5 Mb genomic abnormalities) Inversions Duplications/deletions When to use: As an adjunct to g-banded karyotyping To define complex rearrangements To identify marker chromosomes When publication-quality spectral karyotypes are needed

Μικροσυστοιχίες DNA (DNA microarrays)
Η ηλεκτροφόρηση πηκτώματος διαχωρίζει μόρια DNA και RNA σύμφωνα με το μέγεθός τους. Οι περιοριστικές ενδονουκλεάσες μπορούν να διασπούν τα μόρια DNA σε συγκεκριμένες θέσεις. Η υβριδοποίηση DNA (DNA hybridization) μπορεί να χρησιμοποιηθεί για ταυτοποίηση μορίων DNA. Οι ανιχνευτές υβριδοποίησης (ιχνηθέτες, probes) μπορούν να ταυτοποιήσουν μόρια DNA και RNA που έχουν διαχωριστεί με ηλεκτροφόρηση. Απομόνωση συγκεκριμένων τμημάτων DNA. Η υβριδοποίηση μπορεί να χρησιμοποιηθεί για να ταυτοποιηθεί ένας συγκεκριμένος κλώνος σε μια βιβλιοθήκη DNA (Υβριδοποίηση αποικιών, colony hybridization). Εφαρμογή της υβριδοποίησης DNA στις μικροσυστοιχίες DNA (DNA microarrays).

Μικροσυστοιχίες DNA (DNA microarrays)
Πλέγμα μικροσυστοιχιών (microarray) που συγκρίνει τα μοτίβα έκφρασης δύο ιστών (μυϊκού και νευρικού) στον οργανισμό Caenorhabditis elegans.

Microarrays

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