Nucleic acids hybridization techniques (EXAM 2)

What is nucleic acid hybridization?

Process in which single stranded nucleic acid form different sources but complementary sequences are form double stranded molecules

What do we use nucleic acid hybridization for?

• detect and semi-quantify microbes in a sample

• Localize a microbe in a sample

• Identify a gene in a sample

• Detect gene mutations

• Study gene expression

Probes

A fragment of DNA or RNA of variable length which is used to detect the presence of homologous nucleotide sequences (DNA/RNA target) in a sample

Genomic DNA probe

Fragment (by PCR or DNA cloning, whole genomic (long hundreds to thousands nt)

RNA probes (riboprobes)

By transcription of clones DNA (long hundreds to thousands nt)

Oligonucleotide probes

By chemical synthesis (short 15-50 nucleotides)

Method to labeling probes

By strand synthesis: DNA or RNA template; DNA or RNA polymerase and labeled nucleotide

By end labeling: adding a label to the 5’ end of the probe

Label types

Radioactive, non-radioactive (fluorophores, haptens)

Nick translation - strand synthesis

DNA polymerase removes nucleotides ahead of a nick and replaces them with new nucleotides at the same time

Random primers - strand synthesis

Anneal to multiple locations on a DNA (or RNA derived cDNA) template, providing free 3’-OH ends for DNA polymerase to begin strand synthesis

Labeling with fluorophores and haptens

Fluorophores - direct fluorescent label

Haptens - indirect label detected by antibodies

Specific techniques

South blots, northern blot, dot blot, check board DNA-DNA hybridization, in situ hybridization, DNA microarrays

Who discovered southern blotting and when?

Edward souther -1975

Southern blotting

Uses a DNA probe to detect a sequence in a DNA sample and identify the size of the fragment containing it

Southern blot steps

1. DNA is extracted from cells and cut into fragments by restriction enzymes

2. Fragments are separated by size using gel electrophoresis

3. DNA bands are transferred to a nitrocellulose filter by blotting

4. This produces a nitrocellulose filter w/ DNA fragments positioned exactly on gel

5. Filter is exposed to a radioactively labeled probe for specific gene

6. Filer is then exposed to X-ray film. The fragment contains the gene of interest is identified by a band on the developed film

Who founded northern blotting and when?

James Alwine, 1977

Northern blotting

Uses a DNA or RNA probe to assess presence and quantity of a mRNA in a sample (gene expression)

Dot blotting

Simpler form of Southern and Northern blots: detect and semi-quantitate a DNA or mRNA sequence in a sample without separation (electrophoresis)

Add antibody → detect protein

Add probe → detect DNA

Who invented check board DNA-DNA hybridization and when?

Socransky, 1994

Check board DNA-DNA hybridization

A high throughout version of dot blot used for analysis of dental samples: detection and semi-quantification of up to 40 bacterial species in up to 28 dental samples using whole genomic probes

In situ hybridization

Uses a DNA or RNA probe, usually fluorescent, to detect and localize a DNA or mRNA in a tissue or cells

DNA microarrays

A glass or silicon chip with thousands of unlabeled DNA probes spotted on it for hybridization with labeled nucleic acids from samples

DNA microarrays uses

Detection and quantification of microbes in a sample

Study/compare gene expression

Detect gene mutations (SNPs)

Detect gene copy variations (competitive genomic hybridization)

Microarrays principle

1. Two different cells are being compared

2. mRNA is extracted - the cell’s active gene produce mRNA (more mRNA = gene is being expressed more)

3. mRNA → cDNA - an enzyme called reverse transcriptase converts mRNA into cDNA then cDNA is tagged with a fluorescent label

4. Microarrays chip - each spot contains a DNA probe that matches a specific gene; the labeled cDNA binds to its matching probe through base pairing

5. Scanner reads fluorescence - bright spots mean a lot of cDNA bound (high gene expression); dim spots mean little cDNA bound (low gene expression)