Medicinal Chemistry Final- Fall 2013
Terms in this set (45)
Main tools in recombinant DNA Technology
Plasmid cloning vectors
Extra-chromosomal DNA elements represented by small, circular DNA-- and are present in multiple copies in the cell. Dont use chromosome
Advantages- can carry multiple copies of plasmid
Cloning Vectors must have
Origin of replication
Selective genetic marker-- antibiotic resistance.
How to clone and express a eukaryotic gene in bacteria?
Problems: Eukaryotic genes contain introns which are removed from primary transcript-- exons spliced together before mRNA is exported to cytoplasm--also post-transcriptional modifications (5' capping and polyA tail)
Since the DNA is transformed into bacterial cells which lack splicing apparatus- introns will not be removed and the gene product will not be tranlated (stop codons will likely be encountered by ribosome)
Solution: Don't use genomic DNA- use spliced cytoplasmic mRNA then use reverse transcriptase to synthesize DNA from RNA template.
This DNA synthesized via RT will be cDNA.
Collection of total genomic DNA from a single organism.
DNA is stored in a population of identical vectors, each containing a different insert of DNA.
Cut up genome--->ligate fragments into vector DNA that is cut with restriction enzyme. End result: library of recombinant vectors
To isolate colony with gene of interest
Use Antibodies (western blot, ELISA), direct DNA sequencing, and PCR (use a primer that is designed to bind to specific sequence of DNA that is transcribed)
PCR- polymerase chain reaction
Easy and Fast way to amplify a specific gene from any DNA that contains it.
1. Small amount of template DNA containing segment to be amplified
2. Primers complementary to flanking regions of the DNA segment to be amplified. (2 primers that are complementary to both strands)
3. Thermostable DNA polymerase
4. Nucleoside triphosphates (dATP, dGTP, dCTP, dTTP)
PCR how it works
90 degrees, strands separate
50 degrees, primers anneal
72 degrees, new strands are made (optimal temperature for DNA polymerase)
So you start with single molecule of DNA template then after 10 cycles you will have
2^10 copies of amplified DNA segment
If you start with 4 DNA molecules, after 5 cycles you will have
Collect sample from a patient
run PCR using pathogen specific primers
Mix unknown DNA and all primers in the tube, run PCR, run a dna gel electrophoresis to determine the size of the amplified fragment
Run PCR in multiple tubes each containing a pair of primers specific to one pathogen. The tube with PCR product tell which pathogen is present.
Detection and identification of resistance genes in the pathogen.
PCR with primers specific for antibiotic resistance genes.
Check which of the primer pairs amplified DNA.
Example- primer for B lactamase
To amplify genes expressed as proteins-- RT- PCR
Makes it possible to detect the presence of specific mRNAs in the cell. Isolate total mRNA, anneal primer complementary to mRNA of gene of interest, extend primer with RT making (cDNA), PCR amplification, analyze presence of PCR product.
Real time PCR methods
Faster- possible to detect accumulation of a PCR product without running a gel.
SYBR green- when this dye binds to dsDNA it becomes fluorescent. During PCR as target sequence is amplified SYBR green binds to each new copy of amplified DNA and fluorescence increases
Major disadvantage- Non-specific and can generate false positive signals
Ct parameter (threshold cycle)- number of PCR cycles required to produce a sufficient amount of DNA that can be detected. Ct reflects amount of template DNA in sample, lower Ct, more of the template DNA present.
TaqMan realt-time PCR-
1. TaqMan probe- fluorescent dye at one end and quencher at other. If TaqMan probe anneals to DNA, then during polymerization the Taq DNA polymerase cleaves the probe separating dye and quencher-- fluorescence.
The probe is specific- so a specific oligonucleotide is needed for every target. Good for when specific gene is being tested.
VNTR sequences (variable number of tandem repeats)
microsatellite DNA of repeated sequences and they vary between individuals. Amplify these VNTR sequences using PCR-- applied forensics. Separate fragments by size via gel electrophoresis.
PCR: Gene cloning
Clone DNA with known sequence- is cloned and expressed without the need of using gene libraries.
Total cytoplasmic mRNA-->gene-specific primer and RT--->cDNA plus second gene-specific primer and DNA polymerase--->PCR amplifies specific gene which is cloned into bacteria and expressed.
DNA synthesized from a messenger RNA template in a reaction catalysed by the enzymes reverse transcriptase and DNA polymerase
mRNA+ RT--->cDNA+ DNA polymerase--->DNA amplified and can be recombined into a vector
terminator-based DNA sequencing technology. DNA extension products terminated at different nucleotides will have different colors.
Primer problem- solved by inserting an unknown DNA fragment into the known sequence of the cloning vector. Universal primer
Sequence genome by
1. break up genome into small fragments
2. Sequence individual fragments
3. use a program that assembles short sequences via overlapping sequences to create a
4. cong (contiguous sequence)
Shotgun sequencing using cloning
1. random fragmentation of large DNA
2. clone dna fragments into a vector
3. product is plasmid library
4. plate the clones
5. isolate plasmids from individual clones, sequence inserts, assemble long DNA sequence.
Massively parallel sequencing
Large DNA---> fragmented to DNA fragments--->adaptors with known sequences attached
Flow cell is coated with adaptors that bind to adaptors on DNA fragments.
Primers hybridized to DNA fragments extended by DNA polymerase.
dsDNA denatured and original DNA is discarded leaving newly synthesized DNA that is covalently attached to the flow cell surface.
DNA covalently attached to flow cell via adaptor flips over and hybridized to adjacent primers to form bridge---hybridized primer is extended by DNA polymerase.
Now there is a dsDNA bridge formed which is denatured resulting in two copies of covalently bound ssDNA templates.
This bridge amplification is PCR on a plate that results in clusters of single stranded DNA molecules that form a 'polony'
Each polony represents a cluster of identical DNA sequences that were clonally amplified.
Now---sequencing by synthesis.
The ssDNA fragments are sequenced by hybridizing sequencing primers
Only fluorescently-labeled terminators are added and unincorporated terminators are washed off. The terminator that was incorporated at each polony is determined.
Convert terminator from previous round into 'normal' unlabeled nucleotides and again a fluorescently labeled terminator is added.
Once genome sequenced, needs to be annotated which relies on bioimformatics tools.
Id of protein genes and genes coding for functional RNAs (rRNA, tRNA, etc.)
Prediction of regions involved with regulation of gene expression (promotors, translation initiation sites, etc.)
Assign functions to predicted proteins by comparing proteins encoded in newly sequenced genome with known proteins.
Describe biochemical pathways
ID possible associations of human genome variations with a disease.
Structure of the genome and encoded proteins
Analysis of gene expression and regulation (transcriptome and proteome)
Evolution of genes and genomes, genomes in health and disease
Birth of genomics
Started in 1995 with paper describing 1.8 Megabase sequence of Haemophilus influenze genome
Smallest genomes is of Carsonella rudii- 160,000 bp
Typical genome is 4 million bp and encodes about 4000 different genes.
Bacterial genomes are very densely packed. Many genes are co-expressed as 'operons'
Live attenuated straing of M. bovis
Vaccine developed by passaging a strain of M. Bovis on rich medium 230 times over a 13 year period.
Resulting strain showed reduced virulence but was still immunogenic.
The non virulent BCG- missing 7 gene cluster that are present in MTB. Some of these clusters contain virulence genes (e.g. protein secretion system) which help MTB adapt to human body environment. These virulence genes are not needed in ideal conditions (passaging genes). The attenuated bacteria do not have gene clusters conferring adaptations to human body.
Story 2. Why different BCG vaccines have different efficacy
20th century first half, no way to preserve mycobacteria, strain kept alive via continuous passaging.
By the time BCG vaccine strains stocked world-wide- passed 1500 times.
Different genetic composition from random mutations
Genome analysis showed that different BCG vaccines lack different combos of RD (regions of difference) DNA segments. More lost, reduced potency of vaccine which is related to reduced ability of strain to survive long enough in human body to raise a strong immune response.
Story 3: Genomic helped develop better TB diagnositics
Hypersensitivity rxn from tuberculin injection (mix of TB ag)
Skin test is sensitive, but not specific. A negative skin test rules out TB infection, but positive result does not distinguish between individual infected with MTB or those vaccinated with BCG.
Knowledge of MTB specific regions facilitates development of efficient diag test.
Story 4: Genomics help develop better drugs for TB.
Diarylquinoines (DARQs)- no cross-resistance with known anti-TB agents. So act upon new target.
What is new target?
1. isolated resistant mutants to DARQs of MTB and closely related Mycobacteria.
2. Genomes of wild type and mutant cells sequenced
3. Found mutations that were common in all resistant mutants.
4. Using comparative genomics- Mutations that were common were mapped to atpE encoding ATP synthase.
This finding showed that the DARQ inhibited ATP synthesis.
New AbX targets: essential genes
Inactivate (knock out) as many individual genes as possible and find which genes are dispensable.
AbX resistance markers to gentamicin (for example) using transposons.
Cells that did not receive the transposons die
Cells that do, live.
Cells that confer gentamicin resistance via transposons but die because the transposon disrupted an essential gene
Cells with transposon insertions in non-essential genes grow and form colonies.
From this we know what genes are non-essential and which ones that are.
Ideal ABx targets
Essential- required fro cell survival and proliferation
Ubiquitous- present in all bacteria
Bacteria-specific- not present in humans.
Extract DNA from all bacteria-->prepare PCR primers that recognize two conserved regions-->PCR-amplify DNA
rRNA conserved sequences and VR that are different between individual bacterial species.
Can analyze the number of microbes present by adding up all different variable regions
Can analyze changes in microbiome in response to various factors (change in diet, smoking, drugs, disease).
3 gigabases, about 3 billion bp
Only 1-1.4% encodes proteins (exons)
75% DNA Genes of non-coding RNA (rRNA, tRNA, miRNA, and many other functional RNA species). Regulatory regions: promoters, enhancers, silencers, sequences involved in regulation of chromatin structure, etc.
Almost all of the genome was found to be transcribed into some kind of RNA product
One half of human DNA contains repeated sequences- parasitic. Result of retro-transposition events.
Most abundant repeats are LINES or L1 and Alu repeats or SINES
23,000 protein coding genes.
L1 repeats (LINES)
Long interspersed nucleotide elements
L2 repeat sequence code for gene of RT.
Alu repeats or SINES
Short interspersed nucleotide elements
about 300 bp long.
These mobile repeats affect phenotype through gene inactivation
Transposition using RT into active gene creating inactive gene.
Transcriptome: expressed sequence tags: ESTs
Variety of mRNAs expressed in the cell
Isolate cellular mRNa, clone and sequence cDNA copied from cytoplasmic mRNAs these are ESTs
Transcriptome: DNA microarrays
important application is analysis of identification of disease-related genes.
DNA probes complementary to gene-specific mRNA are prepared and attached to glass slide as an array.
mRNA is prepared from a specific type of cell (cancer cells, specific tissue, etc.) cDNA copy synthesized and labeled with fluorescent dye and hybridized to the chip.
DNA DEEP SEQUENCING REPLACES DNA MICROARRAYS
Expression of many proteins is regulated at level of translation. Therefore transcriptome sometimes does not provide accurate info about gene expression.
Current method to study cell proteome (all of expressed proteins) is via mass spectrometry.
Compare proteome of normal and diseased cells helps understands which proteins good drug targets.
Ribosome profiling likely replace proteomics
The mRNA in the ribosome is being translated. Sequence shows which mRNAs are being expressed.
Distribution of reads between different mRNA shows the relative rates of synthesis of individual proteins. and dist. of reads on mRNA shows how protein is being translated.
Expression of genees in eukaryotic cells regulated at:
Protein folding and stability
siRNA-small interfereing RNA
RNA virus infection- long double stranded RNA formed which is unusual for cell
long dsRNA activates antiviral response
Double stranded RNA cut into short segments by enzyme (DICER). Segments represent ds "siRNA"
ds siRNA associates with protein complex called RNA-induced silencing complex (RISC). Once of strands of siRNA discarded and other remains.
ss RNA in RISC used as address label to target mRNA which base pairs with target in messenger RNS.
viral mRNAs and siRNAs are generated from same segments of viral genome and therefore siRNAs is complementary specifically to virual but not cellular mRNA.
Marks viral mRNA for degradation via RISC
RNAi0 based therpy
Therapeutic precision (target specific gene with no interference of other genes)
Target Destruction- destroys target RNA and prevent production of harmful protein
Problems: stability and delivery
siRNA degrades so replace 2'-OH with 2'F for 2'OMe extend life of siRNA
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