QBM Exam 3
Recall that for dsDNA, an A260 of 1.0 = 50 ng/ul or 50 ug/ml.
-
Determine the ideal annealing temperature given the melting temperatures of two primers.
- the annealing temp in PCR shoudl be ~5 deg. lower than the lowest melting temp (too high prevents binding and too low leads to nonspecific binding)
Recall the key events that lead to the invention of PCR, including some of the key players, developments,and discoveries that lead to its widespread use.
-In 1968, Har Gobind Khorana quadrupled DNA using polymerase and a single primer; involved primer annealing, extension, separation, and re-annealing; not practical -In 1971 a researcher in Khorana's lab, Kjell Kleppe, published a paper where he discussed usin 2 primers. Stuart Linn saw a presentation by Kleppe on the subject, and taught it to students -In 1977 Frederick Sanger used a primer to sequence DNA -Two key developments: Therostable DNA polymerases and automated temp baths -In early 1980's, kary mullis began putting pieces together, no interest from others - in 1984 he was able to amplify DNA and confirm it by SOuthern blot
Describe northern blotting and reverse northern blotting, and when they are most often used.
-Nothern blot invovles searching for RNA with a DNA or RNA probe -Reverse nothern blot uses a labeled RNA probe to search a cDNA library to determine gene expression patterns
Explain the theoretical amplification and limitations of PCR.
-as product increaes, there is competition of complementary daughter strands with primers for reannealing -loss of enzyme activity and oafter ~30 cycles the enzyme concentration are limiting, as too many primers need extending -dNTPs and primers get incorporated every round, and the reaction approaches equilibrium -possible second site primer annealing specificity: primers specific to gene of intrest must be used
List and describe the properties vectors must contain (origin of replication, MCS, and selectable marker).
-be able to replicate in a host oranism, be passed from one genration to the next -be able to confer a selectable phenotype on the host cells -be abel to bring about high-level expression of the passenger gene
List and describe the various protocols that can be used to isolate RNA, highlighting the reagents and columns used along with their purposes.
-cesium chloride gradient -organic solvents anch chatropic salts -detergents liek SDS, N-lauryl sarcosyl, Nonidet P40, etc. -spin column, magentic beads Chomczynski and Sacchi RNA isolation -uses TRIzol which contains phenol, chloroform, and guanidium isothiocyanate to denature proteins and inactivate intracellular RNases
Describe subtractive cloning.
-detect up- and fown-regulated genes -generate DNA an dit complement from cells or an organism, clone what's leftover -cDNA and mRNA, or genomic DNA and a deletion
Describe the forward and reverse primers, what they are made of, and to which strand each anneals.
-foward primer: reads liek gene -reverse primer: inverted -anneals tot he template strand, and the reverse primer to the gene/sense strand -DNA polymerase can use the 3' overhan to copy DNA
Broadly describe the protocol for molecular cloning.
-isolate the gene of interest and generate fragments -insert the gene into a vector to generate a composite molecule of vector + insert -introduce the composite molecule into living cells for propagation =select the cells which contain the gene of intrest -end result: a gene of intrest inserted into a vector, which can be further analyzed or inserted into an expression system or cell
Define the function of DNA microarrays, and list some of its many uses including expression profiling, tumor classification, SNP detection, detecting chromosomal deletions, etc.
-microarrays allow for te screening of thousands of DNA, RNA, and protein molecules at a time -allow for patient-specific treatment, precision medicine
Describe restriction enzymes, highlighting how they evolved, what kind of DNA they recognize, and the different types.
-restriction enzymes cut nucleic acids at specific positions -often recognizes 4,6,8, or 12 bp of DNA -type i/iii: multifunctional, cleave far from (i) or close to (iii) the recognition site, and use ATP and S-adenosyl-L-methionine, and act as methylases -type ii: leave at a specfic site, require magnesium (most useful) -type iv: cleave methylated DNA -type v: use guide RNAs
Compare and contrast blunt vs. staggered/overhang restriction enzyme cleavage.
-some restriction enzymes cut both strands in the center of the recognized sequence, generating a blunt cleavage -some cut at staggered positions on each strand, generating 5' or 3' overhangs (sticky ends)
Recall the process of cDNA library generation.
1. Phenotypic screening: used when the cloned gene is expressed and changes proerties of the cell in an obvious way -antibiotic resistance (surviaval does not gurantee clones will carry the gene of intrest) -blue/white screening -select for mutants or vector markers 2. screening with antibodies 3. DNA hybridazation 4. restriction mapping, PCR, or DNA sequencing
Describe the most common methods of identifying clones, including phenotypic screening, antibody screening, DNA hybridization, and restriction mapping, PCR, and DNA sequencing.
1. antibody screening: -screening with antibodies is used when cloned gene is expressed by E. coli; useful for cDNA library screening 2. DNA hybridiztion: -if part of the nucleotide sequence of gene is known, DNA probes can be generated to identify the clone with the gene of intrest -transfer cells to membrane, lyse, block, hybridize probe to ssDNA devleop, track back to original plate 3. restriction mapping, PCR, DNA sequencing: -the gene of intrest in a plasmid is located by using restriction mapping, PCR, DNA sequencing, or a combination of the three - first, a miniprep is performed, and the plasmid is purified, digested with restriction enzymes, and run on a gel to verify the presence of an insert of the appropriate size
Describe the process of designing primers to introduce tags and restriction enzyme cut sites.
5' overhangs: primers with overhangs can be used to amplify a DNA fragment cut site: allows digestion of PCR product and insertion inot a vector that contains same restriction sites flanking bases: C/G allow polymerase to anneal His tags: six histidines designed into forward primer (CAT or CAC); useful for protein purification via afffinity chromatography
Analyze the relationship between the A260 and A280 of a given sample, and how it relates to DNA purity.
A260/A280 > 1.5 = pure
Describe the RNase protection assay.
An RNase protection assay allows for the sensitive detection and quantification of RNA trasncription levels and to map introns
Compare cloning vectors, shuttle vectors, and expression vectors.
Cloning vectors: -small for increased transformation efficiency, high copy number, many restriction sites, blue/white screening. multiple antibiotic resistance shuttle vectors: -replicates in multiple hosts - the E. coli component of a yeast shuttle vector includes an origin of replication and a selectable number -the yeast component of a yeast shuttle vector includes an autonomously replicating sequences expression vector: -express high amounts of protein from a gene -must insert cDNA in bacteria for protein expression systems -larger, so less efficient transformations, and fewer restriction sites, therefore genes are foten cloned into cloning vectors first, and then digested and ligated into expression vectors
Recall the techniques that involve DNA and RNA hybridization.
DNA hybridization: probe used to find clone in library that contains cloned gene -DNA can be used as probes to detect the presence of their compleemtary sequence. Probes can bring nicks into DNA, add a radio-labeled DNA, add DNA poly, incorporate base into new strand
Compare and contrast the various polymerases including DNA pol I, Klenow fragment, exo-Klenow, Taq, Pfu, and hot start.
DNA pol I: synthesis, porofreading, and primer removal Klenow fragment: polymerization and proofreading, but no primer removal -used to synthesize dsDNA from ssDNA template, remove protruding 3' ends, fin in 5' overhangs after restriction digest, generate radiolabeled probes, PCR prior to Taq exo-klenow: no exonucleae activity taq: very fast but makes ~1 in 9000 mistakes, adds a overhang pfu: slower but makes 1 in a million mistakes, produces blunt ends hot start: polymerases that are inactivated by a aptamer or antibody
Understand the theory and use of agarose gel electrophoresis.
DNA's isoelectric point is ~5, and elctrophoresis is performed at pH 8.3, which ensures DNA has a negative charge DNA has a high charge-to-mass ratio, and migrates logarithmically in the gel primarily on the friction coefficient, which is related to the percentage of the gel, and DNA's size and conformation
Describe and compare the various techniques used to visualize DNA, such as EtBr, SYBR Green, methylene blue, crystal violet, and autoradiography.
EtBr: intercalates DNA and may be mutagenic and carvinogenic -Highly fluroescent when epxosed to UV light only when it inercalates within nucleic acids and thus can be used to detect DNA and dsRNA SYBR Green: methylene blue/crystal violet: allow for real-time visualization of DNA in the gel without the need for developing -not very sensitive, can only detect ~micrograms of DNA autoradiography: detect by exposing to X-ray film -can expose gels ofr day, providing the most sensitive detection
Describe the Southern blot protocol.
In the southern blot, labeled probes can be used to search for a specific DNA sequences in a sample -purify the genomic DNA, digest it using restriction enzymes, separate the fragments using gel electrophoresis, transfer the fragments to a nitrocellulose or nylon membrane, hybridizing a labeled probe to the fragment of intrest, and detecting the probe with autoradiography or fluroscence
Describe and compare Maxam-Gilbert and Sanger sequencing.
Maxam-Gilbert sequencing involves radiolabeling DNA and cleaving it with dimethyl sulfate, piperidine, hydrazine, NaCl sanger sequencing uses a sequence-specific primer and ddNTPs which lack a 3'-OH and terminate synthesis
Broadly describe the patent claims for PCR, along with the court challenges, and eventual ruling.
Patent claims: 1. Purified thermostable Thermus aquaticus DNA polymerase that migrates on a denaturing polyacrylamide gel faster than phosphorylase B and more slowly than does bovine serum albumin and has an estimated olecular weight of 86k to 92.5k 2. the polymerase of claim 1 that si isolated from therus aquaticus 3. the polymerase of claim 1 that is isolated from a recombinant organism transformed with a vector that codes for the expression of thermus aquaticus DNA polymerase Court challenges: -Dr. Kornberg, stated that the actions by Hoffman-LaRoche to restrain the use and extension of PCR technology by universities and nonprofit research institutions "violated practices and prnciples basic to the advancements of knowledge for the public welfare" PCR Patent Ruling: -false information was presented to the courts in order to recieve the patents
Compare and contrast RFLP and VNTR, highlighting their uses.
RFLP: involves analyzing SNPs, which chnge the restriction enzymes digestion site and thus pattern on a southern blot VNTR: involves analyzing STRs, which are highly variable in th epopulation -both can be used for pedigree and inheritance analysis, disease diagnosis, as geneitc markers in mapping genes, paternity testing, tissue matching, genetic fingerprinting, and in forensics
Describe RNase, including where it can be found, how to inhibit it, and how to inactivate it.
RNase A is a single-strand specific endoribonuclease -found in cells and tissues in high amounts -also on hands to fight pathogens ans in miniprep reagents -resistant to metal chelating agents -can survive prolonged boiling and autoclaving RNase activity is extremely difficult to inactivate -add inhibitors or denaturing agents such as B-ME, diethyl pyrocarbonate, guanidium isothiocynate, or recombinat ribonuclease inhibitor
Compare reporter/hydrolysis probes and SYBR green dye.
Reporter/Hydrolysis Probe: -In the 5' nuclease assay, an oligonucleotide probe with a reporter/quencher at each end is added to the PCR mixture -the probe anneals to a specific sequence of template between the forwards and reverse primers -when the probe is intact, it will emit light from the quencher, but if cleaved, it will emit light from the reporter SYBR green dye: -nonspecific qPCR is frequently performed using SYBR green die to its versatility -SYBR green emits a fluorescent signal when it binds to dsDNA, which increases as more dsDNA amplicons are produced -cost-effective and convenient, but SYBR green dye will bind to any dsDNA molecule, so it is not specific -partial solution is a melt curve: slowly increase the temperature to detect dissociations. A pure samplewill have one peak, impure samples have multiple peaks, primer dimers have very low Tm
Define TA cloning and Ligation Independent Cloning.
TA cloning: TAQ adds As to PCR products, ligate directly to vector that has T overhangs ligation-independent cloning: uses primer sequences that are compemtary to a linearized plsmid; directionality guranteed; direct cloning of pCR products w/o ligation
List the reagents used in PCR, along with their purposes.
TAQ polymerase: creates complementary pairs to ssDNA; prone to error but fast and survives high temp two ssDNA primers (forward and reverse): targets the DNA of intrest for aplification dNTPs: nucleotides used by DNA polymerase to copy DNA Magnesium: catalyzes the reaction and stabilizes dNYP pyrophosphate leaving group Tris buffer (pH 8.5-9): keeps pH constant so TAQ polyerase can function Pfu: reduces error rate but slow
Explain the mechanism of DNA ligase.
The enzyme ovalently attaches AMP to a 5'-phosphate of a DNA fragment, which is then attackedby the 3'-OH from a second frgment, forming a phosphodiester bond between the two strands and releasing AMP
Explain the protocol necessary to use two restriction enzymes that are functional only under different buffer conditions.
The fragment must br digested with one enzyme, run on a gel to remove the buffer, cut out via gel extraction to remove the target DNA from unwantef fragments, and digested with the second enzyme
Given an image of a gel, approximate the number of base pairs represented by a band compared to markers.
The migration distance of fragments in the gel will be inversely proportional to the log of the number of base pairs
Describe which of the above techniques are combined in a miniprep.
alkaline lysis and silica-based beads
Define the theory behind Sanger sequencing, and the role ddNTPs play.
allowed fro sequencing to be performed in a single tube
Briefly describe the various genomics PCR techniques, including asymmetric PCR, anchored PCR, and genome walking, asymmetric PCR, anchored PCR, genome walking, and inverse PCR, solid-phase PCR, helicase-dependent PCR, transcription-mediated amplification, aptamers, and isothermal amplification.
asymmetric PCR: one primer at limiting concentrations to generate ssDNA probes anchored PCR: amplify targets when only one end is known; chromosome walking inverse PCR: digest target, circularize, linearize to inverse, PCR target solid-phase PCR: PCR directly on beads or in oil; bridge or emulsion PCR helicase-dependent PCR: PCR at room temp, potential for handheld devices
Synthesize expected results of a recombinant DNA experiment, determining the effect of inserting a vector into cells with/without an insert, IPTG, X-gal, etc. (similar to the book and homework questions).
blue: LacZ intact - B-galactosidase - X-Gal cleaved -blue if LacZ gene in MCS is intact (took up vector but did not take up gene of interest) white: LacZ disrupted - B-galactosidase - no X-Gal cleaved -white if vector and gene of intrest was taken up inot MCS, disrupting LacZ gene, and with addition of IPTG
Understand why RNA extraction and purification can be challenging for researchers.
can be challenging due to RNA stability - RNA is more suseptible to degradation than DNA, due to the 2 hydroxy group adjacent to the phosphodiester linkages in RNA -mRNA 5' cap polyA tail hold off degradation for a short time -DNases require metal ions for activity and can be inactivated with chelating agents but RNase do not use metal ions
Define and compare the various types of mutants including conditional, conditional lethal, and non-conditional, recalling examples of each.
conditional mutants: can grow under one set of conditions (permissive) but cannot grow under different conditions (restrictive or nonpermissive) -ex: temperature-sensitive mutants and bacteriostatic antibiotics and resistance conditional letgal mutant: can grow under one set of conditions (permissive) but dies under different conditions (restrictive or nonpermissive) -ex: bactericidal antibiotics and resistance, or other compounds liek 5-FOA; attaching part of beta-lactamase to the gene of intrest to restore resistance; a restriction enzyme that digest genomic DNA if present non-conditional or biochemical mutants: display the mutant phenotype under all ontidions, require a supplemnt or enzyme to survive (typically bacteria or yeast auxotrophs) -ex: B834 Met E. coli require methionine and URA3 S. cerevisiae require uracil
Define and compare the various DNA purification techniques including crude lysate, salting-out, anion exchange, cesium chloride gradient, phenol-chloroform extraction, alkaline lysis, and silica-based methods.
crude lysate: heat cells, add proteniase K (not very clean) salting-out or precipitation using potassium acetate, centrifugation, alcohol (used in other protocols) anion exchange: backbone binds to + charged beads, alcohol precipitation cesium chloride gradient: high purity, but time-consuming phenol-cholorform extraction: phenol (aqeuous, contains DNA and RNA) Chloroform (organic, contains proteins and cellular debris), isoamyl alcohol alkaline lysis: SDS: breaks up phospholipid bilayer NaOH: dissolves structural proteins agitation, precipitation, centrifugation silica-based methods: selective adsorption of DNA to silia-based gels in the presence of chaotropic salts
Describe the steps of a standard PCR reaction.
denaturing: dsDNA into ssDNA at high temperature annealing: attaching primer to each piece of ssDNA extension: elongation of primers by DNA polymerase at 3' end, creating new dsDNA
Define star activity and its causes.
enzymes may cut nonspecifically under certain conditions
Define bisulfite sequencing and RNA-seq, listing examples where each are used.
epigenetic analysis can be performed using various methods, such as bisulfite sequencing (deaminating cytosine to uracil) -samples can be analyzed with PCR or sequencing RNA-Seq uses next generation sequencing to determine the RNA present in a tissue or single cell -replacing microarrays for expresion profiling
Given a strand of DNA, design forward and reverse primers to amplify the sequence (similar to the book and homework questions).
forward: identical to the 5' end sequence of gene reverse: reverse complement to the 3' sequence of gene
Define and compare genomic, cDNA, and electronic libraries.
genomic library: all of the DNA in the cell, fragments - gene library approach: clon the entire genome and find what you need -can use bacteria, phage, or YACs -genes can be transcribed and translated, but will not form a functional protein (screen with DNA probe) cDNA library: all of the expressed genes, without introns -eukaryotic genes with introns can be hundreds of thousands of base pairs, which make clonin difficult, and introns can account for 95% of a eukaryotic gene -bacteria do not have a spliceome, so cDNA must be used for protein expression systems -starting from mRNA, a cDNA library can be generated, which represents protein-coding genes - can be transcribed into a functional protein (screen with a DNA probe or antibody) electronic library: digital library based on the human genome project
Explain why the E. coli cell lines used in the lab are unique and beneficial for research experiments.
highly efficient, grow well, easy to regulate, take up foreign DNA, no proteases, restriction enzymes, or recombination, allow for many plasmids, high/loe mutation rates
Analyze a restriction enzyme buffer table, and determine what buffers should be used for two different restriction enzymes.
if digestin with two enzymes, they must wither be compatible (>50%) or you must digest with one, run a gel, perform a gel extraction, and digest with the second
Recognize and analyze graphs of qPCR results, including amplification plots and melt curves.
melt curve: -raise temp of qPCR products slowly and measure change in fluorescence as DNA dissociates -pure DNA: single peak at 80-90 C -multiple peaks indicate multiple samples of DNA -Determine purity, analyze differences in sequences Ct threshold: line chosen to compare samples to etermine relative amount of starting material in the original samples
Briefly describe the various genotyping PCR techniques, including genotyping, colony PCR, touchdown PCR, nested PCR, multiplex PCR, AFLP PCR, and RAPD PCR.
multuplex PCR: multiple primers for multiple targets touchdown PCR: Start at a high annealing temp and lower it; increase specfiity nested PCR: Use nested internal primers to increase specificity gene typing: identify an organism by bands produced via PCR AFLP: like RFLP, add adapters with cut sites, PCR from them RAPD: random primers to amplify differences individuals
Describe the various methods to induce competency in cells, including natural, chemical, electroporation, and phage packaging.
natural: some strians of bacteria (not E. coli) are naturally able to take up DNA under certain consitions -demonstrated that DNA is the genetic material chemical: cells can be treated with cheicals that partially disrupt the cell membrane, rendering it permeable to added DNA molecules -E. coli cells can be treated with CaCl2 or other chemicals electroporation: DNA can be introduced into cells with an elctric current -cell membrane is breifly disrupted, DNA enters, and the membrane reforms -can be used with eukaryotic cells -fast, and provides a time constant, but more expensive in vitro packaging:DNA can be packaged in lamda phage particles and nitroduced into bacterial cells in vitro -an internal 20 kb of phage DNA can be replaced with a gene of intrest or human genomic DNA -the rDNA will packaged in phage particles and mixed with E. coli and plated. The phage will infect the bacteria and ake man copies of the gene. Viral plaques are areas of many cloned phage carrying the gene of interest
Compare traditional and next-generation sequencing.
next-generation sequencing: recent advances in DNA sequencing and allows for millions od bp to be sequenced in a single run -several newer techniques add specific bases to a DNA template and measure synthesis
Differentiate between the different types of vectors including plasmid, bacteriophage lambda, phagemids and cosmids, plastids, BACS and YACS, and viral vectors, recalling examples when each would be used.
plasmids: bacterial cells may contain extrachromsomal DNA called plasmids that can carry antibiotic resistance genes that are shared via conjunction ex: used in bacteria like E. coli bacteriopage lamda: historicaly one of the most important developments in molecular cloning due to theri ability to hold ~5x the DNA of plasmids ex: used to clone large DNA fragments in E. coli phagemids and cosmids: --many plasmids have an f1 phage origin that allows for packaging in phage (phagemids), requires a pilus. still limited to ~4000 bp of DNA but can allow for more plasmid copies as if its not under bacterial control -cosmids are a combination of a plasmid and labda phage, has phage cos sites for packaging, and can clone up to 40,000 bp of DNA. some can replicate in eukaryotes as well ex: can replicate in multiple hosts Ti plasmids and plastid DNA : -Transgenic plants are produced to provide growth enhancements, disease resistance, and herbcide resistance by using bacteria to insert the gene into the plant's nuclear genome ex: used to replicate multiple hosts Bacterial (BACs) and Yeast artifical CHromosomes (YACs) can be used for cloning millions of base pairs -BAC: up to 300,000 bp, easier to work with, transformed via electroporation -YAC: up to 3 million bp, more difficult Viral vectors are used to introduce new o ealtered genes into the genomes of humans and animal cells
Describe fluorescence in situ hybridization (FISH), recalling examples of when it is used.
position of DNA on chromosome can be identified
Describe the different types of next-generation sequencing including microarrays, oligos, mass spec, pyrosequencing, Illumina, ion semiconductor, nanopore, etc.
pyroseqencing: can sequence bases by binding small fragments of DNA to beads immersed in oil and amplifying them by emulsion illumina (solexa) sequencing: uses bridge PCR to link primers to a solid support medium; base added detected by fluorescence ion semiconductor: works by adding dNTPs and detecting changes in pH nanopore: involves passing a DNA sequence through a small nanopore and measuring the change in current shape
Compare and contrast traditional PCR and qPCR.
qPCR measures the kinetics of the reaction in the early phases of PCR, providin a distinct advantage over traditional pCR detection -simultaneously amplify and quantify DNA, without the need to run an agarose gel three phases: -exponential: exact doubling of product is accumlating at every cycle and the reaction is very specific and precise -linear (high variabaility): the reaction component are being consumed and the reaction is slowing as it approaches equilibrium -plateau (end point): the reaction has stopped, no more products are being made -traditional PCR measures in the plateau phase, where as qPCR measures in the exponential phase - some forms of qPCR can comfirm which gene is being amplified
Describe the specificity of qPCR, explaining the theory behind the technique.
qPCR using a reporter probe is extremely specific, and reports only amplification of the target (even if other products are amplified) Taq polymerase's 5'-3' exonuclease removes any downstream DNA, indicating PCR has occured in real time
Given a gene sequenced by Sanger sequencing, determine the sequence of either the primer or the gene (similar to the book and homework questions).
read from bottom, then reverse complement
Define RT-PCR and RT-qPCR.
reverse transcription PCR (RT-PCR) can amplify RNA into a DNA product, which can be analyzed on a gel -similar to a northern blot, but much higher sensitivity RT-qPCR allows quantification of RNA in a sample or tissue using qpCR -essential technique in many laboratories, and has replaced the northern blot and ELISA for sample analysis and detection of cancer, infectious diseases, and gentic disorders -requires less sample than northern, can amplify from a singel cell and detect femtograms, no need fro gel,probe,blot, etc. -more sensitive than ELSIA, can amplify 1 viral particle per microliter in less than one hour and catch infections early
Define the dot blot and explain its limitations in comparison to the Southern blot.
see if NDA is present or not, tells nothing abouit size -equivalent to ELISA (antibody searching for proteins) but this is a probe searching for DNA -unlike southern blot, dot blot can only tell you the size of band is hybridized to
Briefly describe the various recent PCR developments, including solid-phase PCR, isothermal amplification, helicase-dependent PCR, transcription-mediated amplification, and aptamers.
solid-phase PCR: Emulsion PCR and bridge PCR, performed in oil or on beads -primers can be linked directly to beads, so each bead contains pCR products that are clones of the same DNA, and physically linked to the beads for easy retrieval isothermal amplification: -can be performed at room temp, without a thermo cycler -helicase-dependent PCR can separate dsDNA by breaking the hydrogen bonds, primers anneal, polymerase extends -loop-mediated isothermal amplificaion uses 4-6 primers to form aggregates of dsDNA =rapid detection of pathogens -much faster, but requires optimization, more template DNA transcription-mediated amplification: -isothermal amplification -used to test blood for HIV
Define the relationship between restriction enzyme specificity and the length of the recognition site.
specificty increases directly with length of recognition site
Describe how sticky ends can be converted into blunt ends.
sticky ends can be converted to blunt ends by Klenow or T4
Given an image of a gel, determine what conformation DNA is in for specified bands, such as nicked, circular, linear, and supercoiled.
supercoiled DNA travels the fastest, followed by linear, cirvcular, and nicked
Compare and contrast agarose gel electrophoresis and polyacrylamide gel electrophoresis (SDS-PAGE).
the higher the agarose gel %, the smaller the pore size, and therefore the gels have a better resolution fro small DNA fragments and vice versa
Understand how DNA fragments of different sizes move on an agarose gel.
the lower the DNA's size, the greater the distance traveled
Calculate the melting temperatures of various primers using the formula Tm= (A+T)x2 + (G+C)x4.
the melting temp is the point at which 50% of the DNA is bound, and it is important to know for the annealing step
Describe the function of topoisomerase, alkaline phosphatase, T4 polynucleotide kinase, and reverse transcriptase.
topoisomerase: relax supercoiled DNA alkaline phosphatase: removes 5' and 3' phosphates from DNA to prevent ligation T4 polynucleotide kinase: phosphorylates DNA at 5' for ligation, probes, removes 3' phosphate reverse transcriptase: generates cDNA from mRNA
Determine the protocol necessary to ensure proper insert directionality.
two different restriction enzymes are used in cloning to guarantee insert directionality two different restriction enzymes, at least one should generate a staggered cut, while the other can also genrate a staggerd cut or a blunt cut
Recall and define the various tools researchers use in recombinant DNA technology, such as vectors, restriction enzymes, DNA polymerase, ligase, reverse transcriptase, ribonucleases, etc.
vectors: vehicle used to carry DNA sequene into a host cell for cloning restriction enzymes: DNA-cutting enzymes responsible fro bacteriophage restriction DNA polymerase: copies and repairs DNA, and function as an exonuclease, DNA polymerase uses a template strand to synthesize new DNA in the 5-3 direction ligase: joins two matching pieces of DNA together reverse transcriptase: making a copy of DNA from RNA ribonucleases: transferase that catalyzes the hydrolysis of ribonucleic acid deoxynucleases: DNA primase: an enzyme that synthesizes short RNA sequences called primers topoisomerase: relieves tension during DNA replication by breaking the phosphodiester backbone of DNA, passing one strand across another, and reforming the bond gel elctrophoresis: laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size clones: identical copies of a single original cell
