CSB349H1 Lecture Notes - Lecture 1: Reverse Transcriptase, Orf 2, Hybridization Probe
14 May 2018
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DNA Retrotransposons – LTR
(long terminal repeats)
DNA Transposons
Lecture 1(c): Genome Repeats
Fluorescence in-situ Hybridization (FISH):
• Allows to measure the copy number (# of repeats) in DNA – interspersed repeats
• Generate a DNA probe synthesized by PCR with fluorescently labeled nucleotides
o Probe will be an anti-sense copy of the repeat sequence in interest
(anti-sense does not have to be the entire sequence; only short portion is required)
§ Probes are hybridized to fixed cells or tissues
ð Also works if we denature chromosomes and add a large amount of the
DNA probe which will eventually base-pair with the complementary
denatured chromosome
o This is followed by washing step, to wash off the probes that are
not hybridized to the chromosome and then observed under a
fluorescent microscope
§ # of fluorescent tags = # of copies
Transposable Elements (ex. of interspersed repeats):
1. DNA Transposons:
o Cut themselves out of the genome and re-insert themselves
in another region
§ “cut and paste mechanism”
i. Transposon code for an enzyme: transposase which
functions to:
• Make blunt-ended cuts in donor DNA
• Make staggered cuts in target DNA
ii. Transposase then functions to ligates the donor DNA
into the target DNA
• Cellular mechanisms recognize there are unpaired
ssDNA and functions to ligate back together
2. DNA Retrotransposons:
o Transcribe DNA into RNA (RNA intermediate), which is
later reverse transcribed back into DNA
§ Newly synthesized DNA sequence is re-inserted
into another region
i. DNA is transcribed by RNAPII to generate an mRNA
ii. mRNA is transported into the cytoplasm and is
translated protein; encode for reverse transcriptase
iii. Reverse transcriptase will enter the nucleus and convert
mRNA back into DNA
• cDNA is inserted back into the genome via
integrases (coded by the protein region)
ð Retrotransposons are very similar to RNA retroviruses:
o RNA produced is not packaged & released; instead the cDNA is integrated in genome
Document Summary
Probes are hybridized to fixed cells or tissues. Also works if we denature chromosomes and add a large amount of the. Dna probe which will eventually base-pair with the complementary denatured chromosome: this is followed by washing step, to wash off the probes that are not hybridized to the chromosome and then observed under a fluorescent microscope. # of fluorescent tags = # of copies. Transposable elements (ex. of interspersed repeats): dna transposons, cut themselves out of the genome and re-insert themselves in another region. Dna transposons: transcribe dna into rna (rna intermediate), which is later reverse transcribed back into dna. Retrotransposons are very similar to rna retroviruses: Dna retrotransposons ltr (long terminal repeats: rna produced is not packaged & released; instead the cdna is integrated in genome. Long interspersed elements mechanism of transposition: lines are non-ltr elements, lines are highly abundant elements in our genome, replicate themselves and position themselves inside our genome; are known to being.
