Genomics And DNA Technology 5 Flashcards
Elements of human genome
- only about 2% are coding
- genes about 20%
- 50:50 unique vs repeated sequences
- genome smaller than your microbiome (bacterial sequences)
- mitochondria also have a a genome (maternal)
- genome encodes 21,000 proteins, but also RNAs and 3D formation that regulate gene expression
- genome sequence can be used to predict your health and drug response
- complexity is more correlated to gene number than genome size
Genome variability
- protein coding genes (open reading frames, ORFS)
- sequence comparison reveals many homologous genes as well as horizontal gene transfer (bacteria obtain DNA from environment in same time period)
- genome sequence variability is 0.1%
- Single nucleotide polymorphism (SNPs) (point mutation with 1 nucleotide change)
- copy number variation (VNTRs)
- insertion, deletions, translocations (big regions of DNA)
- mutations are rare, approx 60 per person but dont often change expression
Types of mutations
-point mutations can be:
-silent (no amino acid change)
-missense (different aa)
-nonsense (premature stop codon)
Frameshift mutations: addition or deletion of nucleotides (other than multiples of 3) changes translation reading frame
-splice site mutations: incorrectly processed RNA… intron not removed
-trinucleotide repeat expansions: tandem repeats of codons (Huntington’s disease, Fragile X syndrome, Myotonic dystrophy)
Chromatin modifications and epigenetics
- epigenetic: heritable information NOT contained in the DNA sequence
- histone modification: (e.g. Acetylation decreases histone interaction with DNA and promotes transcription)
- DNA methylation: inhibits transcription
- affects how easily DNA wraps
epigenetics controls gene expression
- gene switched on
- active (open) chromatin
- unmethylated cytosines
- acetylated histone
- gene switched off
- silent (condensed) chromatin
- methylated cytosines
- deacetylated histone
-environment of fetus affects epigenetics
Complementary nature of DNA is the basis of molecular medicine
- site specific digestion of DNA with restriction endonucleases (proteins that cut DNA)
- amplification and expressions of DNA by cloning into vectors
- amplification of DNA between specific primers using PCR
- creation of genomic libraries in a vector collection
- DNA sequencing
- Probe hybridization to determine genotype (SNPS, Southern Blot) or gene expression (microarray, northern blot)
- Manipulation of genes and gene expressions (RNA, CRISPR_
Restriction enzymes and gene cloning
- DNA of interest is cleaved by Taqi
- Second fragment of DNA with sticky end produced by taqi
- 2 cohesive ends form hydrogen bonds
- DNA lipase forms a 3’ to 5’ phosphodiester bond between fragments to reform DNA
Polymerase chain reaction (PCR)
- Denature DNA into separate strands (heat)
- Anneal primers to flanking regions of single stranded DNA (cool…)
- Extend primers with DNA polymerase (use heat resistant enzyme +Mg) 72 degrees so enzyme doesnt denature
- The 2 new double stranded DNA molecules can be denatures and copied again
DNA libraries
- libraries are large collections of DNA fragments, cloned into a vector, from which individual sequences can be selected and isolated for characterization
- genomic libraries contain genomic DNA clone fragments
- complete genome (sequencing)
- cDNA clones in cDNA library
- expressed genes only (expression)
Traditional DNA sequencing
-Sanger sequencing uses 4 chain terminating nucleotide analogs and gel electrophoresis to generate a DNA sequence “ladder”
“Next generation” DNA sequencing
- DNA polymerase incorporates a complementary nucleotide into the new DNA chain and unreacted nucleotides are washed away
- the incorporated nucleotide is detected by its fluorescence, then the fluorescent in the group is removed
- A new solution of nucleotides is introduced
Eg. Illumina sequencing: shorter read length but sequences analyzed in parallel
DNA Probes
- single stranded DNA probes (oligonucleotide) can be used to detect the presence and/or amount of a specific DND or RNA sequence in a sample
- fluorescent DNA binds to a target probe
- sensitive to as small as a single base pair mutation
- genomic or expression probes
- can be single base pair specific
- genomic probes can detect the presence of sequence variations due to mutations, single nucleotide polymorphisms (SNPs) and variably repeated sequences (VNTRs), microsatellites
- another approach amplifies via PCR then runs on gel
- FBI combined DNA index system
Micro arrays
- use multiple oligonucleotide probes arrayed at known locations on a 2D surface to detect the presence or amount of complementary DNA in a sample
- use for both comparative gene expression analysis and high density genotype gentlemen such as SNPs analysis
CRISPR
- clustered regularly interspace short palindromic repeats: adaptive immunity in bacteria
- short guide RNAs from past infection are stored and used to speak out matching DNA sequences: hybridization results in double strand cleavage by enzyme Cas9
- can be used to delete a gene, or to replace/edit cleaved gene
Genome
-complete set of genes of an organism
