Gene technology and molecular diagnostics
Hoofdstuk 1: Introduction
1.1 Genome
Genome = complete set of genetic material present in a cell or organism
- Functional genome analysis unravel the role of genes within those genomes
• Transcriptome analysis = study the expression of genes or gene sets
• Proteome analysis = find out which proteins are synthesized
• Interactome analysis = discover how all these molecular interactions are linked
1.1.1 Prokaryotic genomes
- Circular genome
- Plasmids
• Can replicate independently from the chromosome
OR be integrated in larger chromosome (as an episome) and replicate together
with chromosome
• Can tranfer from one bacterium to another through conjugation
1.1.2 Eukaryotic genomes
- C-value paradox = overall genome size is not related to the complexity of the organism
- DNA wrapped around histone proteins → chromatin
- Multiple linear chromosomes in the nucleus
- DNA in organelles (prokaryotic origin)
• Mitochondria
• Chloroplasts
- Repetitive DNA
1. Tandem repeats = satellite-DNA
• Mini/micro/macro satellite DNA (micro = short tandem repeats)
• A certain motive is repeated for a number of times
2. Dispersed repeats = transposable elements
• Jumping DNA
• inverted repeat sequences at its ends
• DNA transposons
o Transpose through conservative transposition = cut and paste
o Transpose through replicative transposition = copy and paste
o Enzyme that execute excision = transposase
1
, • Retro-elements or retrostransposons
o Jump via RNA-intermediate (copy-and paste)
o Type retro-elements:
1. LTR = long terminal repeat
2. non LTR = LINES (long interspersed nuclear elements) / SINES
(short interspersed nuclear elements)
o From RNA-intermediate → DNA via reverse transcriptase
New genetic variants can be formed through:
- Homologous recombination (cross-overs)
- Non-homologous recombination
• Translocations
• Non homologous end joining: repair after dsDNA break
- Small nucleotide changes in the genome
• Mutations
• Deletions
• Duplications
• Insertions
Multi-gene families = group of similar genes with similar sequence and function
- Members of family = homologous genes
• In the same species : paralogues
• In different species: orthologues
- Function
• Need for a lot of gene product (vb histones)
• Functional diversification of the family members
• Sometimes family members loose their function = evolution of pseudogenes
Horizontal gene transfer (HGT) = Lateral gene transfer (LGT)
= process in which an organism incorporates DNA from another unelated organism into its own
genome
- How to detect HGT:
• Look for presence of a gene in species/genus that is not present in related
species/genera but is very similar to a gene from an unrelated species
- Vb. Conjugation between bacteria, between organelles and nuclear DNA
2
,1.2 Gene expression
Gene = a part of the DNA that encodes an RNA molecule
1.2.1 In prokaryotes
Gene codes for a single protein via an Open Reading Frame (ORF)
- ORF= from ATG till stopcodon
1.2.2 In eukaryotes
- Gene contains exons and introns → need to be removed via splicing
- One gene can code for several proteins via alternative splicing
- In normal circumstances gene inactive
• DNA wrapped into chromatine ( = heterochromatine)
o When activated + compact: euchromatine
• Eukaryotes have a lot of genes (to much energy to keep them on all the time)
• Eukaryotes are multicellular (not all gene products required in every cel)
→ transcription factors needed to activate
- Transcription
• RNA polymerase = enzyme that synthesizes RNA from a DNA template
• Regulated by the binding of transcription factors to the promoter region
o Promotor = sequence of DNA to which proteins bind to initiate
transcription
o Binding sites of promotor = enhancer regions = cis-acting/regulatory
elements
- After translation: protein localization
• Proteins have signal peptides that guide them to the target site
1.3 Transcriptome
= set of all RNA transcripts
- mRNA –- reverse transcriptase enzyme → cDNA (= DNA copy)
- primer used as starting point: oligo-dT primer (eukaryotes), gene-specific primer or
random primer
Non coding RNA: many transcripts from genome that are not translated
1.4 Levels of gene regulation
1.5 Basic techniques for DNA-analysis
1.5.1 Gel electrophoresis
DNA molecules can be separated by size in a gel
- DNA is negative and moves to positive pole
- Larger molecules move slower
- DNA ladder = mixture of DNA fragments of known lengths used as size reference
3
, Agarose gel <-> PAGE = poly acrylamide gel electrophoresis
1.5.2 Restriction enzymes
= enzymes that cut double stranded DNA at specific recognition sites or restriction sites
→ creates restriction fragments
1.6 Basic principles recombinant DNA
Recombinant DNA = artificial new combinations of different genes or DNA fragments
Vb. Production of human insulin in bacteria
- Insert something in a vector using DNA ligase
• cDNA
• part of genome
• PCR product
• Restriction fragment
• Combination of the above
- Vector
• Replicating unit that can be opened to insert another DNA fragment
• Can multiply inside host cell, independently of the host cell genome
• Lot of vectors are man-made plasmids
• Different vectors for different host cells available
DNA replication
- Hydrogen bonds between bases break → strands separate
- Each DNA strand is template and determines the sequence of bases in the new strand =
complementary base pairing rules
- Enzyme for DNA synthesis = DNA polymerase
- 5’ → 3’
4
Hoofdstuk 1: Introduction
1.1 Genome
Genome = complete set of genetic material present in a cell or organism
- Functional genome analysis unravel the role of genes within those genomes
• Transcriptome analysis = study the expression of genes or gene sets
• Proteome analysis = find out which proteins are synthesized
• Interactome analysis = discover how all these molecular interactions are linked
1.1.1 Prokaryotic genomes
- Circular genome
- Plasmids
• Can replicate independently from the chromosome
OR be integrated in larger chromosome (as an episome) and replicate together
with chromosome
• Can tranfer from one bacterium to another through conjugation
1.1.2 Eukaryotic genomes
- C-value paradox = overall genome size is not related to the complexity of the organism
- DNA wrapped around histone proteins → chromatin
- Multiple linear chromosomes in the nucleus
- DNA in organelles (prokaryotic origin)
• Mitochondria
• Chloroplasts
- Repetitive DNA
1. Tandem repeats = satellite-DNA
• Mini/micro/macro satellite DNA (micro = short tandem repeats)
• A certain motive is repeated for a number of times
2. Dispersed repeats = transposable elements
• Jumping DNA
• inverted repeat sequences at its ends
• DNA transposons
o Transpose through conservative transposition = cut and paste
o Transpose through replicative transposition = copy and paste
o Enzyme that execute excision = transposase
1
, • Retro-elements or retrostransposons
o Jump via RNA-intermediate (copy-and paste)
o Type retro-elements:
1. LTR = long terminal repeat
2. non LTR = LINES (long interspersed nuclear elements) / SINES
(short interspersed nuclear elements)
o From RNA-intermediate → DNA via reverse transcriptase
New genetic variants can be formed through:
- Homologous recombination (cross-overs)
- Non-homologous recombination
• Translocations
• Non homologous end joining: repair after dsDNA break
- Small nucleotide changes in the genome
• Mutations
• Deletions
• Duplications
• Insertions
Multi-gene families = group of similar genes with similar sequence and function
- Members of family = homologous genes
• In the same species : paralogues
• In different species: orthologues
- Function
• Need for a lot of gene product (vb histones)
• Functional diversification of the family members
• Sometimes family members loose their function = evolution of pseudogenes
Horizontal gene transfer (HGT) = Lateral gene transfer (LGT)
= process in which an organism incorporates DNA from another unelated organism into its own
genome
- How to detect HGT:
• Look for presence of a gene in species/genus that is not present in related
species/genera but is very similar to a gene from an unrelated species
- Vb. Conjugation between bacteria, between organelles and nuclear DNA
2
,1.2 Gene expression
Gene = a part of the DNA that encodes an RNA molecule
1.2.1 In prokaryotes
Gene codes for a single protein via an Open Reading Frame (ORF)
- ORF= from ATG till stopcodon
1.2.2 In eukaryotes
- Gene contains exons and introns → need to be removed via splicing
- One gene can code for several proteins via alternative splicing
- In normal circumstances gene inactive
• DNA wrapped into chromatine ( = heterochromatine)
o When activated + compact: euchromatine
• Eukaryotes have a lot of genes (to much energy to keep them on all the time)
• Eukaryotes are multicellular (not all gene products required in every cel)
→ transcription factors needed to activate
- Transcription
• RNA polymerase = enzyme that synthesizes RNA from a DNA template
• Regulated by the binding of transcription factors to the promoter region
o Promotor = sequence of DNA to which proteins bind to initiate
transcription
o Binding sites of promotor = enhancer regions = cis-acting/regulatory
elements
- After translation: protein localization
• Proteins have signal peptides that guide them to the target site
1.3 Transcriptome
= set of all RNA transcripts
- mRNA –- reverse transcriptase enzyme → cDNA (= DNA copy)
- primer used as starting point: oligo-dT primer (eukaryotes), gene-specific primer or
random primer
Non coding RNA: many transcripts from genome that are not translated
1.4 Levels of gene regulation
1.5 Basic techniques for DNA-analysis
1.5.1 Gel electrophoresis
DNA molecules can be separated by size in a gel
- DNA is negative and moves to positive pole
- Larger molecules move slower
- DNA ladder = mixture of DNA fragments of known lengths used as size reference
3
, Agarose gel <-> PAGE = poly acrylamide gel electrophoresis
1.5.2 Restriction enzymes
= enzymes that cut double stranded DNA at specific recognition sites or restriction sites
→ creates restriction fragments
1.6 Basic principles recombinant DNA
Recombinant DNA = artificial new combinations of different genes or DNA fragments
Vb. Production of human insulin in bacteria
- Insert something in a vector using DNA ligase
• cDNA
• part of genome
• PCR product
• Restriction fragment
• Combination of the above
- Vector
• Replicating unit that can be opened to insert another DNA fragment
• Can multiply inside host cell, independently of the host cell genome
• Lot of vectors are man-made plasmids
• Different vectors for different host cells available
DNA replication
- Hydrogen bonds between bases break → strands separate
- Each DNA strand is template and determines the sequence of bases in the new strand =
complementary base pairing rules
- Enzyme for DNA synthesis = DNA polymerase
- 5’ → 3’
4
