Flashcards in Ch 5 Deck (74):
act as precise scissors for cutting specific DNA sequences
allows the separation and identification of specific proteis and nucleic acids using gel electrophoresis
the genome sequences of entire organisms can be determined
solid-phase synthesis of nucleic acids
specific sequences of nucleic acids can be synthesized and used to identify or amplfy other nucleic acids
polymerase chain reaction pcr
allows the billion fold amplification of DNA to obtain sufficient quantities for further characterization
how are restriction enzyme names deriived?
three letter abbreviation for the host organsim followed by strain designation and a roman numeral
how do restriction enzymes provide a fingerprint?
pattern of fragments produced by a restriction enzyme can also serve as a fingerprint of that specific DNA molecule
what is used to separate bands cut by restriction enzymes by size?
how is the restriction digest pattern dna visualized?
stained with ethidium bromide and using UV light
how do you identify an unknown restriction fragment on a gel?
describe southern blotting
agrose gele is transfered to a nitrocellulose sheet and washed with a solution containing radioactive complementary dna sequene. then audioradiography shows where the dna probe is located
how is are RNA sequences on a gel identified?
how is a protein on a gel visualized?
what is the sanger dideoxy method?
invented by frederick sanger: based on the generation of DNA fragments whose length is determined by the identity of the last base int he sequence using controled termination of replication: done using dideoxy analogs of each NTP
how is the sequence read after sanger dideoxy method?
red from the pattern of chain termination. each column shows fragments ending in a different base
what is a more commonly used sequencing reagent nowadays?
in what direction does DNA synthesis occur?
3' to 5'
how is dna synthesized?
chemical syntehsis by sequention addition of activated monomers
what is the size limitation of dna synthesis
about 100 bp
how are larger fragments of dna synthesized?
multiple dnas syntehsized coresponding to a much larger sequence and joined to form new tailr-made genes
what are uses for dna synthesis?
short DNAS used in pcr, and dna probes for blotting techniques
what must be known for PCR? and what needs to be synthesized before PCR?
flanking sequences of target DNA must be known. primers that are complementary to flanking sequences must be synthesized before pcr
what is required for pcr?
pair of primers that hybridize and are complementary to flanking sequences of dna target
all for dntps
a heat stable dna polymerase from thermophilic bacteria
thermal cycler : machine that cycles between different temperatures
a single PCR cycle ocnsists of three steps:
1. strand separation: two strands of target dna molecules are separated by heating at 95 C
2. hybridization of primers
solution is quickly cooled to 54 C to allow primers to hybridize to the 5' and 3' ends of target dna
3. dna synthesis: solution is heated to 72 C which is optimal temperature for dna syntehsis by taq DNA polymerase (taq is from thermus acquaticus)
how many times is the PCR cycle repeated? and how can you calculate amiplifaction from this number?
20-35 times. amplification is 2^n fold
(1 million fold after 20 cycles, 1 billion fold after 30 cycles)
what are applications of PCR?
medical diagnostics (very small amount of bacgterial and viral (hiv) can be detected using pcr
forensics (dna from crime scene can be greatly amplified for further identification)
DNA from extinct orgnanisms can be amplified for evolutionary studies (neanderthal genome)
mediwhat does DNA ligase do?
catalyzes joinng of 2 dna duplexes having compatible overhangs
what does DNA ligase require/
a free 3' hydroxyl group nd a 5' phosphoryl group. both DNAs must be double helical
energy source such as ATP required for joining the DNAs
what is dna ligase used for?
can be used to insert novel dna sequences into a dna vector
what are two commonly used vectors
plasmids and phages
how are vectors prepared for cloning?
cutting vector with a suitable restriction enzyme followed by ligation with target DNA. both vector and dna target must have compatible ends
what is a dna vector?
it is inserted into a host where it replicates autonomously
what are plasmids
plasmids are circular, double stranded dna molecules that occur naturally in some bacteria: cary genes for a selectable marker such as antibiotic resistance. contain a site that tolerates insertion of a new dna sequence
what do plasmids contain? (puc18)
1.origin of replication required for propagation in the host organism
2. ampicillin resistance selectable maker
3. beta-galactosidease gene encodes a protein that breaks down a sugar analog to produce a blue color
4. beta-galactosidase gene containsa polylinker sequence containing many restriction sites
how does the presense of an insertion affect the betagalactosedase?
disrupts the beta-galactosidase gene and produces a white color
what are phages?
phages are viruses that infect bacterial cells and replicate (bacteriophages) inject dna into bacterial cell resulting in the production of more viral particles
what are the 2 modes of infection
1. lytic pathway
2. lysogenic pathway
what is the lytic pthway
- viral functionsare fully expressed
-leads to destruction of the hos cell and relase of hundreds of virus particles
hat is the lysogenic pathway
the phage DNA is integrated into the host genome and can be replicated together with the host DNA
Why would lambda phages be used as cloning vectors?
phages can tolerate larger DNA insertionsthan plasmids. the modified viruses enter bacteria much more easily than plasmid vectors
how is a genomic library created?
1. genomic dna is first digested into large fragments
2. fragments that are about 15kb long are isolaed using gel electrophoresis
3. these fragments are ligated to lambda dna using compatible ends
4. ecoli bacteria are infected with these recombinant phages
5. phages replicate and lyse or kill their bacterial hosts
6. resulting lysate contains a large number of phage particles containing fragments from entire genome => genomic library
how is the genomic library screend to find ag ene of interest?
can use a hybridization with a radioactive dna probe.
1. ecoli bactera plated into a lawn
2. phage infects this bacterial lawn giving rise to clear plaque
3. a replica of this plate is made using nitrocelluose paper
dna on paper is denatured with sodium hydroxide and hybridized to a radioactive probe
how are probes designed?
using mrna extracted from cells. probe can also be designed of part of protein is known
what are the 3 types of mutagenesis?
deletions, substitutions, insertions
how are deletions made?
aspecific sequence within a larger dna can be excited using restriction enzymes and the remaining ends can be joined by dna ligase
or you can use overlap extension pcr
what is overlap extension pcr?
uce pcr to make targeted deltions of any size: primers for first round of pcr have single stranded extensions that are complimentary to each other
what is site directed mutagenesis
noble prize 1993 michael smith. mutant proteins made containing a single amino acid substutition using oligonucletodies (primers) with desired mutation. mutant primer annelaed to dna using dna and elongated using dna polymerase. original parent dna is degraded using DpnI which cleaves methylated dna
how are insertions made?
castte mutagenesis involves cutting plasmid dna with 2 different restrictio nenzymes to remove a specific region. a newly synthesized dna fragmant containing compaitble ends is then ligated into paldmi.
how are genes synthesized?
many oligonucleotides are synthesized which correspond to the desired sequence. the 40-100 base oligonucleotides are annealed and joined together to form the final dna sequence for the protein. final sequence is clonsed into a plasmid for protein expression
what is the shotgun method to sequencing dna?
shatter genomic dna into many smaller pieces followed by sequencing these fragments which were then analyzed for overlapping regions which determines how they come together to form full genome sequence
how many base pairs, genes, and chromosomes does the human genome have?
3 billion base pairs, 24 chromosomes, 25000 genes
what accounts for a very complex proteome?
alternative splicing and post translational modification
how much of the genome codes for proteins and how much does for RNA
1.5% codes for proteins
> 90% is transcribed into rna at high levels
what insites does comparative genomics lead to?
1. allows for the identification of novel genes
- comparison of the human and pufferfish genomes lead to the identification of 1000 previously unknown genes
2. evoluiontary relationships can be determined
- comparisons between human, chimpanzee, and neanderthal genomes gives insights into our own evolutionary history
the complete genome sequence allows us to systematically look at the expression levels of all the individual genes in an organism. what assumption is this based on?
based on assumption that levels of mRNA indicate the level of protein being produced in the cell
how do gene chips help to analyze gene expression?
high density arrays of oligonucleotides can be constructed which are complemetary to the mRNAs produced by various genes
binding of mRNA extract to dna microarray (or gene chip) results in fluorescence
fluorescence quantified for gene expression
why must one start with mrna to isolate gene for expression? what happens to the mRNA?
if its from eukarytoic dna, it will have intron sequences that are removed only after mRnA is expressed. mrna is converted to dna using reverse transcription.. resulting cDNA is then ligated into a protein expression plasmid
how do you produce cDNA
1. synthetic oligo(dT) primer is annealed to the poly(A) of the mRNA
2. reverse transcriptase uses the free 3'-OH end to initiate cDNa synthesis
3. treatment with alkali (sodium hydroxide) at high pH is used to degrade the RNA strand
4. terminal transferase is used to add a string of dGs to the 3' end of the newly synthesized cDNA to create another primer site of known sequence
5. PCR is then used to amplify the cDNA using the oligo(dT) and olicgo(dC) primers
how do protein expression vectors work?
1. cDNA is inserted into plasmid directly after a plasmid encoded transcription promotor
2. a ribosome binding site is located just before the start codon of the gene to be expressed (cDNA)
3. the resulting cDNA clones can be screened for expression of the protein of interest
why do eukaryotic hosts sometimes have to be used for expression of a target gene?
bacteria lack enzymes required for post translational modification of eukaryotic proteins such as the addition of carbohydrate groups on the surface of proteins
bacteria also lack the chaperone proteins that assist proper folding of proteins in eukaryotic cells
some eukaryotic proteins need these functionalitites in order to be expressed
what are 3 ways recombinant genes can be introduced into eukaryotes/
1. microinjection, 2. electroporation, 3. viral vectors
what is microinjection?
dna is directly injected into the nucleus of a cell using a micropipette
what is electroporation?
a high voltage pulse is used to make the cell membrane permeable to DNA molecules
how are viral vectors used?
retroviruses can integrate dna version of their rna genomes into the hosts chromosomal dna, which can then be expressed by host cell machinery
what are the most efficient vectors for delivery of foreign dna into eukarytoic cells? and up to how many kb inserts can they accept?
retro viruses, 6kb
how are gene knockouts made?
made using homologous recombination with a mutant version of the gene.
1. a mutant version of the garget gene is designed
2. the mutant maintains some similarity with the WT especially at the 5' and 3' ends
3. when this gene is introduced into embryonic cells, recombination occurs between similar regious leading to the replacement of the WT gene with the inactive mutant version
4. look for phenotypic effects on organism
how can dsrna be introduced into c. elegans worms?
directly feed them e. coli bacteria tat produces dsrna
what does introduction of dsRNA do?
disrupts the mRNA from genes that contains sequence corresponding to dsRNA molecule
how does double stranded rna cleave mRNA?
dsRNA is cut by dicer into 21 nucleotide fragments which have 2 nt of unpaired base at the 5' ened. the 2 strands of these fragments are separated and incorporated into the risc complex. the single stranded 21nt RNA serves to guide RISC to a complementary mRNA which is then degraded
what are three ways recombinant genes can be introduced into plants?
1.tumor inducing plasmids
-integrate into genome and can express foreign dna
-use high voltage electrical pulse makes the cell wall permeable to dna
- dna is coated onto tungsten pellets and tehn fired into plant cells at high velocity
what are the effects of agrobacterium tumefaciens infection of plants
common soil bacterium agrobacterium tumefaciens infects plants and introdeucies foreign dna
a tumor (crown gall) grows at sight of infection
crown galls syntehsize opine which are metabolized by bacteria
the metabolism of plant cell is diverted to produce food for agrobacterium
how do tumor inducing plasmids work?
tumor inducing plasmids carried by agrobacterium are responsible for the shift to tumor state and synthesis of opines
a small portion of the Ti plasmid called T-dna is integrated into the palnt cell genome
foreign dna can be inserted into the tdna region and expressed upon infection into a plant
only works with dicots (broad-leaved plants such as grapes) and some monocots
how does electroporation work in plants?
cellulose wall is first degraded by treatment with cellulase to form protoplasts
a mixture of plasmid dna and protoplasts is subjected to high voltage electrical pulses
dna enters cells and expresses foreign DNA