CHAPTER 5 DNA HYBRIDIZATION (cont.) - GEL ELECTROPHORESIS (basic concept) -SOUTHERN, NORTHERN, WESTERN BLOTTING - In situ HYBRIDIZATION -MICROARRAYS -

CHAPTER 5

DNA HYBRIDIZATION(cont.)

- GEL ELECTROPHORESIS (basic concept)- SOUTHERN, NORTHERN, WESTERN BLOTTING

- In situ HYBRIDIZATION- MICROARRAYS- RFLP ANALYSIS

MISS NUR SHALENA SOFIAN

DNA Gel Electrophoresis• DNA is negatively charges (because of phosphate backbone)• DNA will be attracted to positively charged poles and

repelled from negatively charged ones

Basic method:• Molten agarose poured into casting tray; a comb is inserted• After agarose solifies, comb is removed leaving wells where

DNA will be loaded• DNA samples mixed with tracking dye (sucrose/glucose to

weigh down the DNA; dyes to track the migration of DNA)• A buffer containing ions (to conduct electric current) is

placed in the chamber tank

DNA Gel Electrophoresis

Movement of DNA fragments in agarose gels

• There is a linear relationship between the migration rate of a given DNA fragment and the logarithm of its size (in bp)

• Larger molecules move more slowly through the gel because of friction

Visualizing DNA

• EtBr- a flourescent dye visualized when excited by UV light- intercalates into DNA molecules, thereby ‘staining’ it

• Gel is soaked in solution of EtBr and the DNA bands take up the dye

• Gel is placed under UV light and visualized/photographed

An EtBr-stained gel photographed under UV light

a. DNA electrophoresis sample loading b. DNA bands visualized under UV transilluminator

SOUTHERN BLOTTING(Edward Southern, 1975)

• Questions: How can you find a specific piece of DNA in a mixture containing many DNA molecules?

• Separate DNA molecules according to their sizes – variations of gel electrophoresis can separate large DNA chromosomal sized DNA molecules as well as DNA molecules that differ by a single nucleotide

• Gel electrophoresis can be done under conditions that preserve native DNA structure or denature DNA molecules

• DNA molecules separated by size using gel electrophoresis can be transferred to a support matrix (e.g. nitrocellulose membrane) and hybridized to radioactive probe (e.g. 32P- labeled DNA)

• This technique is called Southern Blotting

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Northern Blotting• Question: How actively a corresponding gene is

transcribed in a number of different tissues of an organism?

• Similar concept to Southern blotting, except one examines RNA instead of DNA

• RNA is isolated from particular cell type under certain conditions or during development stage

• Probes can be either RNA or DNA • The conditions for RNA-RNA or RNA-DNA

hybridization are somewhat different than the conditions for DNA-DNA hybridization

• RNAs that complement to radiolabeled probe are detected as dark bands on X-ray film

• E.g. detecting tropomyosin protein (responsible in muscle contraction) would reveal in smooth muscle cells and striated muscle cells but not in brain cells (due to absence of DNA bands)

Northern Blot techniqueThis figure shows a developmental northern blot used to investigate the expression of the Pax6 protein in the mammalian embryo. Pax6 is critical for normal eye development; mutations in the Pax6 gene result in small eyes (in heterozygous mice) or no eyes or nose (in mice or humans homozygous for the loss-of-function mutation). The northern blot shows that this gene is expressed in the embryo in the brain, eyes, and pancreas, but in no other tissue.

Northern Blot analysis

Northern-blot analysis of tissue distribution of PSAT-specific mRNAMultiple-tissue Northern-blot membranes (MTNTM I and II; ClonTech Laboratories), each containing 2 µg of poly(A)+ RNA/lane, were hybridized with 32P-labelled PSATa, PHGDH (phosphoglycerate dehydrogenase) and β-actin cDNA probe.

Western Blotting

• Analyzes protein instead of DNA or RNA• The basic process is the same as Southern and

Nothern but differ in probes used• One can use antibody specific to a particular

protein to identify it on the blot

Methods:• Disrupt protein in detergent, coating it with

negative charges and separates via SDS-PAGE• Blotting the protein with nitrocellulose membrane• Using antibody as probe to recognize protein of

interest – primary antibody for that protein• An antibody binds to epitopes (3D structure) of an

antigen • An unbound primary antibody is washed away• Secondary antibody (radiolabeled/ conjugated to

an enzyme) is added to bind to the primary antibody – provides a way to detect protein of interest in gel blot

Western Blot: Sample Preparation

Western Blot technique

Western Blot technique

In Situ HYBRIDIZATION

• Labeled probes used to hybridized to chromosomes – locating the gene of interest

• Chromosomes from certain cell is spread, DNA is partially degraded revealing ssDNA so it can hybridize/complement to probe

• X-ray film to detect the label – darkening of photographic emulsion locates the labeled probe

• Microscope fluorescent – fluorescent molecule absorbs light at particular wavelength and emits light at longer wavelength

• This technique can also uses fluorescently labeled DNA probe – FISH

• Incorporating biotin-labeled nucleotides into the probe to make it fluorescent

• Other common fluorescent dyes used are DAPI (4’, 6- diamino-2-phenyl-indol)

• Applications: in biology, genetics research and clinical – detecting small changes in chromosome structure e.g deletions, duplications which lead to genetic disorders

FISHFluorescent in situ hybridization (FISH) was used to detect replicating DNA (yellow) of an adenoviral vector in 293 cells. This vector carries the gene for factor VIII and was detected with a labeled FVIII cDNA probe. Nuclei had been counterstained with propidium iodide (red).

Cytogenetical mapping of the sex-determining region (SD) of medaka. FISH of metaphase chromosomes. SL2(sex-linked marker 2, red) localizes on the short arms of the sex chromosomes, whereas a BAC clone containing SL1 (sex-linked marker 1, yellow) localizes on the long arms of the sex chromosomes.

DNA Microarrays (Gene Chip)• Slide made from silica, plastic, or glass that has been covered

with small dots of known genetic markers - ~500 and several thousand bp, and 'printed' onto the slide

• DNA is prepared with fluorescent markers attached to the external frame, and cut into segments appropriate for the testing taking place

• DNA is then layered onto the surface of the microarray, where complementing strands will bond with the markers attached to the slide

• Strands may show some variance in their ability to bind; some will completely bind, some will not bind, and some will partially attach

• The microarray is then rinsed in a buffer solution to remove any strands of marked DNA that have not bonded to a site on the slide; distinct pattern of phosphorescent and blank sites on the surface of the chip

• To read the results, a scanning laser microscope is used to check each bonding site in many small portions, or pixels, from which an average fluorescence is calculated. This number is then used to quantify the level of expression of whichever sequence the site coded for by corroborating with a map of the sites' relative locations

Applications

• Cell-specific gene expression• Gene regulation• Elucidation of metabolic pathways• Tumor profiling• Genetic variation• Microbial strain identification• DNA-protein binding

Microarrays

Microarrays

Each spot on the array corresponds to a specific gene. Colour of the spots anlyzed via computer imaging techniques, indicates amount of RNA transcribed from that gene

Restriction Fragment Length Polymorphism (RFLP)

• One place where Southern blotting is useful is the characterization of differences in DNA sequence between individuals

• One type of study is RFLPs• Remember that restriction enzymes cut DNA at

specific sequences• And although our genes may be very closely

related, there are differences in the sequence of all of our DNAs (except for identicial twins)

• As our DNAs vary, it naturally follows that the restriction maps of pieces of our DNA will vary as well. Many of these differences are inconsequential for gene function. Others may be linked to genetic diseases or disorders

• Polymorphism means genetic locus that has different forms or alleles.

• This simply means cutting up DNA from any two individuals with a restriction enzyme yielding fragments of different lengths

DNA Fingerprinting using VNTR’s

• A special kind of RFLP analysis is called DNA fingerprinting. It relies on the analysis of what is called as Variable Number of Tandem Repeats (VNTR)

• VNTR’s consists of many tandem repeats of a short sequence - minisatellites

• A particular person’s VNTR comes from genetic information donated by the parents; VNTR can be inherited from a mother or father, or combination of both, but never a VNTR either of the parents do not have

• The repeats vary from individual to individual depending on the amount of crossing over that occurs between related sequences

• Since sequences are repeated, there are many places when crossing over can occur

• If it is unequal, two VNTR sequences of the same size will give rise to bigger VNTR sequence, the sum of whose sizes is the same as twice as the original size of the VNTR

DNA Fingerprinting using VNTR

• On some human chromosomes, a short sequence of DNA has been repeated a number of times

• In any particular chromosomes the repeat number may vary from one to thirty repeats

• Since these repeat regions are usually bounded by specific restriction enzyme sites, it is possible to cut out segment of the chromosome containing VNTR

• Run the total DNA on a gel, identify VNTR by hybridization with a probe specific for the DNA sequence of repeat

• A family consists of a mom and dad, two daughters and two sons. • One daughter is from the mother’s previous marriage, and one son is adopted • After amplifying the VNTR DNA from each member of the family, it is cut with a restriction enzyme and run on an agarose gel• Daughter 2 is the child from the mother’s previous marriage and son 2 is adopted.

Application of DNA Fingerprinting in Forensic

• Question: How can you tell a Ghanaian boy is a son to a British mother?

• Almost all individuals have different DNA patterns inherited as in Mendelian fashion e.g. dominant, recessive, heterozygote

• DNA fingerprinting has potential in identfying criminals• Using probes that hybridize to a single DNA locus that

varies from one individual to another rather than a whole set of DNA loci

• Each probe gives simpler patterns containing one or few bands

Case study: Detecting a rapist by DNA typing

• Two possible suspects for the same crime• Take a blood sample from each person and "run" a gel electrophoresis, comparing their DNA with any blood found at the crime scene• Look for the suspects' DNA fingerprints match the fingerprint of blood from the crime scene.

QUESTION:According to the gel, which suspect is guilty, based on the DNA found at the crime scene?