Microbiome Techniques Lecture 7

what is nucleic acid hybridization?

Process in which single stranded nucleic acid from different sources but complementary sequences form double stranded molecules (separate and put together again--single strand from one source + single strand from another source but complementary to one another)

nucleic acid hybridization occurs with...

➢ ssDNA with ssDNA

➢ ssDNA with ssRNA

➢ ssRNA with ssRNA

process of nucleic acid hybridization

native state -denaturation-> single stranded denatured state -renaturation-> renatured state

simplified process of nucleic acid hybridization

denature, see if they complement each other, rehybridize (annealing)

if strands bind and there is a signal, it is ______. if there is no signal, it is _______

positive, negative

uses of nucleic acid hybridization

❑ Detect and semi-quantify microbes in a sample

❑ Localize a microbe in a sample

❑ Identify a gene in a sample (e.g. resistance gene).

❑ Detect gene mutations

❑ Study gene expression. (how much is a GOI expressed?)

what is a probe?

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

probe types

genomic DNA probes, RNA probes (riboprobes), oligonucleotide probes

which probe type is for long nucleotide sequences?

genomic DNA, RNA (hundreds to thousands of nt)

which probe type is for short nucleotide sequences?

oligonucleotides (15-50 nucleotides)

genomic DNA probes

can be either

➢ Fragment (by PCR or DNA cloning)

➢ Whole genomic (nonspecific)

RNA probes (riboprobes)

➢ By transcription of cloned DNA

oligonucleotide probes

➢ By chemical synthesis (sequence sent to company)

methods of probe labeling

strand synthesis, end-labeling

strand synthesis

DNA or RNA template; DNA or RNA polymerase and labeled nucleotide

end labeling

adding a label to the 5' end of the probe

label types

radioactive and non-radioactive

radioactive labels

frequently used

non-radioactive labels

fluorophores

haptens-biotin and digoxigenin

strand synthesis - nick translation

DNAse I's exonuclease/ polymerase domains create a nick, removing nucleotides and adding new ones that are labeled/ complementary to that strand. this fragment is the probe of interest

strand synthesis - random primers

random primers bind to RNA templates, adding new labeled nucleotides, amplification with PCR

strand synthesis - riboprobes

make a new strand in form of RNA - linearize strand to make vector flat/ linear, then add RNA polymerase

hapten definition

when attached to large carrier molecule acts as "tag" recognized by specific antibodies

fluorophore/ hapten methods

could be conjugated to enzyme that changes color once substrate is added; using a fluorophore to see a signal from the sample

southern blotting

Restriction enzyme breaks GOI into fragments, uses a DNA probe to detect a sequence in a DNA sample and identify the size of the fragment containing it--cross-links UV light exposure, fixes DNA to membrane

southern blot-

probe type:

target type:

sample type:

detects?:

needs electrophoresis?:

details?:

probe type: DNA

target type: DNA

sample type: DNA

detects?: presence, specific sequence, size of fragment sequence

needs electrophoresis?: yes

details?: add basic pH to help strands denature, expose filter to UV light overnight on nylon membrane

northern blotting

Uses a DNA or RNA probe to assess presence and quantity of a mRNA in a sample (gene expression). Based on intensity of signal you can tell if it's low/ high expression

northern blot-

probe type:

target type:

sample type:

detects?:

needs electrophoresis?:

details?:

probe type: DNA/ RNA

target type: mRNA

sample type:

detects?: presence, quantity, gene expression

needs electrophoresis?: yes

details?: gel needs formaldehyde to denature RNA, uses restriction enzyme, nylon membrane

dot blotting

Simpler form of Southern and Northern blots: Detect and semi-quantitate a DNA or mRNA sequence in a sample without separation (electrophoresis)--extract DNA/ mRNA, place samples on membrane, add probe to detect and quantify

dot blot-

probe type:

target type:

sample type:

detects?:

needs electrophoresis?:

details?:

probe type: DNA or mRNA

target type: DNA or RNA sequence

sample type:

detects?: presence/ semiquantity

needs electrophoresis?: no

details?: simple/ quicker, nitrocellulose membrane

checkboard DNA-DNA hybridization

A high throughput 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. Instead of using blots, uses "lines"

checkboard

probe type:

target type:

sample type:

detects?:

needs electrophoresis?:

details?:

probe type: whole genome

target type: bacterial species

sample type: dental

detects?: presence/ semiquantity

needs electrophoresis?: no

details?: multiple types of bacteria simaltaneously, nylon membrane

in situ hybdrization

Uses a DNA or RNA probe, usually fluorescent, to detect and localize a DNA or mRNA in a tissue or cells instead of membrane. Denature DNA in a tissue and add probe

in situ-

probe type:

target type:

sample type:

detects?:

needs electrophoresis?:

details?:

probe type: DNA/ RNA (fluorescent)

target type: DNA or mRNA

sample type: tissue in cell

detects?: presence and location

needs electrophoresis?: no

details?: ONLY way to localize samples in tissues without membrane

Fluorescent in situ hybridization (FISH)

add probe and allow it to hybridize in situ, use heat to denature tissue to allow it to hybridize, shows if positive and where it is located on tissue

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-

probe type:

target type:

sample type:

detects?:

needs electrophoresis?:

details?:

probe type: labeled DNA

target type: labeled nucleic acid (DNA or RNA)

sample type: labeled nucleic acid (DNA or RNA)

detects?: presence/ quantification/ gene expression (RNA)

needs electrophoresis?:

details?: glass/ silicon chip, requires colors to tell us what is going on

what is special about DNA microarrays?

sample rather than probe is labeled

uses of DNA microarrays

❑ Detection and quantification of microbes in a sample

❑ Study/compare gene expression

❑ Detect gene mutations (SNPs)

❑ Detect gene copy variations (competitive genomic hybridization)

principle of DNA microarrays

one bacteria treated with antibiotic, one is not. They then bind to complementary genes on chip. Green-control, red-found in gene, yellow-both, black-neither

example of DNA microarray

Human Oral Microbiome Identification Microarray (HOMIM): Sample hybridized simultaneously against probes to 300 different species per slide. Indicates what bacteria is in oral microbiome