Unit 9 Flashcards
Define site directed mutagenesis.
Method to introduce single residue mutations into an expressed protein based on restriction sites in gene of interest.Gene can be cleaved at restriction site and an a segment that is identical except a few bp changes can be spliced in based on the same restriction cleavage.
Define oligonucleotide directed mutagenesis.
- A primer (oligonucleotide, 21-40 bp long) is generated complementary to the sequence to be replaced except with the desired bp changes. Complementary primer also created.- Gene is denatured with primers and annealed.- Primer provides 3’ site for DNAp. DNAp synthesizes new whole plasmid from template and primer.- Original primers may be degraded by DpnII if selectively methylated or otherwise marked. Mutant plasmids may be ligated and anneal to form mutant cDNA.
Distinguish oligonucleotide directed mutagenesis from site directed.
Oligo uses primers to prompt creation of a whole new plasmid from the cDNA template. Site directed depends on restriction sites around mutation point in order to replace the original, non mutant DNA.
Describe how DNA primers and probes of a specific sequence are synthesized using solid phase synthesis.
- Solid phase or silicon beads are in column.- Blocked dNTPs have a DTP, acid labile blocking agent attached to its 5’ end and are fixed by 3’ end to silicon.- Acid washed through removes block.- Next base to be added: blocked dNTP of that base is washed through column. Excess removed.- Acid removed… repeat.- Finally, cleavage rxn removes cyanohydrin protective groups and cleaves polypeptide product from silica.
Suppose you want to use oligonucleotide directed mutagenesis to make a mutant gene that encodes a protein containing phenylalanine rather than tyrosine. Given the following DNA and protein sequence, write the sequence of the primers you would design to make the mutant protein….. Cys Ser Leu Tyr14 Gln Leu Glu……. TGT AGT CTC TAT CAA TTA GAA…
Original:…. TGT AGT CTC TAT CAA TTA GAA…New: ……..ACA TCA GAG (phe) GTT AAT CTT ….Different possible sequences that are an anticodon for Phe can be put in.
Describe a procedure used to tag proteins thereby facilitating their purification using affinity chromatography techniques.
- Make a fusion protein gene (restriction enzyme fusion of separate genes) with a specific, well characterized affinity tag on either of the terminalsEx: His tag, GST- Tag gene fused to protein gene- Expressed via expression vector- Lysate collected and the tag can be used to isolate, evaluate presence, or see interactions of the tagged proteins based on reversible binding interactions with a precipitate or solid state bound ligand
What is needed in order to amplify a segment of DNA using PCR?
1) DNA segment from which the sequence will be copied - the template2) Two oligonucleotide primers that will anneal to ends of the sequence, directed towards the inside of the sequence in the 3’ direction3) dNTPs that will be used to synthesize the new DNA4) DNA polymerase - will synthesize the new strands and should be a heat shock resistant strain
Discuss the three repeated steps PCR: Heating, Cooling, and Replication.
1) Heating - denatures DNA and primers2) Cooling - anneals primers to DNA3) Replication - with mild heating, DNAp (Taq) binds to primers and extends from 3’ ends inwards.Repeating the cycle = exponential amplification of the primer flanked sequence/gene of interest
PCR: For each copy of the target sequence that you started with, how many copies do you expect after cycle 10 (assuming that the amplification performs perfectly in each cycle)?
After round three, starting with 2 copies of endproduct, you will get 2^(#rounds total-3) copies per each original DNA strand.Ex: 2(10-3) = 2^7 = 128 copies
Explain why the use of DNA polymerase from the bacterium Thermus aquaticus (found in hot springs) made a big improvement for PCR.
The bacteria polymerase is used to operating at elevated temperatures and most importantly, will not denature following each round of heating to denature the double stranded DNA. If the DNAp were not heat resistant, the DNAp would need to be replaced following every round.
What is an STR? Discuss the use of PCR as a forensic tool.
A short tandem repeat sequence: conserved between people and have similarly conserved flanking sequences. However, STRs are different lengths for different people.- DNA fingerprinting: amplifying a set of known STRs via PCR and then measuring the lengths of the products gives rise to a profile unique to each person
Describe how PCR could be used to detect HIV infection at an early stage, including what information about HIV would be needed to begin with.
- qPCR can be used to amplify any HIV DNA that is produced by immune cells due to the infection- Degree of infection/amount of virus DNA present can be on the rate at which the anti-HIV gene binding-labile probe is activated in qPCR- This diagnostic test requires that you know at least end sequences of the gene coding for part of the virus which you can base your primers on
Do problems 5 and 8 on pp. 353 - 354.
Primer 1) for complement strand = the same base sequence as the __ bp before the beginning of the gene in the known strand or sequence you are provided withPrimer 2) for the known strand = the complement base pair sequence for __ bp after the end of the gene of interest (complement to the provided strand, oriented so the 3’ primer end is facing towards the inside of the gene)
What does RT-PCR sand for and why is it used?
Reverse Transcription PCR.Used to amplify the cDNA that corresponds to an isolated mRNA sample. Good for:- Determining how protein expression (proportional to mRNA production) varies under certain conditions or (in combination with qPCR) at different time points following an alteration
What does qPCR stand for and why is it used? Describe how qPCR works.
Quantitative PCR.Used to measure relative amounts of a DNA present in an original sample from the organism genome or from a cDNA sample.- Gene of interest will bind complementarily with a fluorescent probe that is binding labile and activated.- The greater the presence of the replicated gene of interest, the more fluorescence activated.- The quantification = the rate at which the fluorescence at a specific wavelength reaches a threshold intensity as measured by absorption spectroscopy.
What is the meaning of the term “library” to a molecular biologist?
Library refers to a collection of cloned DNA from a sample that can be used to study the functions of the genes
What is a genomic library?
A collection of cloned DNA from the entire organism genome. Largest type of DNA library.
What is a cDNA library?
A collection of DNA copies of all the mRNA being produced in the organism sample at a specific time or under specific conditions.
Describe the synthesis of cDNA.
Lysate is mixed with poly-T primers that anneal to poly-A 3’ tail of mRNA that has been post processed in sample cells (no introns). Reverse transcriptase binds and produces DNA from RNA. RNA is degraded by base. DNA is primed from 5’ end by oligo nucleotide known for small segment of the gene. DNA p synthesizes other DNA strand.
Describe the synthesis of a genomic library.
A collection of cloned DNA from the entire organism genome. Genome is partially digested, (correct size segments) inserted into large segment vectors (BACs, YACs) and cloned. Each cell grows into a colony of clones all producing that same cloned segment of DNA.Largest type of DNA library.
In preparing a genomic library for a given kind of animal or plant, does it matter from which type of cell you prepare the DNA? Why or why not?
Genomic library generation depends on restriction sites to cleave genome. If common or well conserved sites are used for the partial digestion, it should not matter. All organisms use DNA, the universal code for genome coding.
In preparing a cDNA library, does it matter from which type of cell you prepare the mRNA?
Yes, it does matter, because some cells have constitutively transcribed genes (housekeeping genes) which must be negatively regulated, while others only produce mRNA for genes when positive regulation induces transcription. A eukaryote may have a smaller cDNA library at a given time because of this. Even more specifically, different factors in different tissues will influence the mRNA production and can give very different cDNA libraries for different parts of the same organism (inherent in specialized cells)
How would a cDNA library prepared from mouse liver cells differ from a cDNA library prepared from mouse thyroid cells?
Would be secreting and producing different proteins (different non constitutive mRNAs) suited to different function. Thyroid - secretion of hormones, etc. Liver - secretion of digestive and nutrient processing enzymes.
How do the short sequences generated using reversible termination sequencing assemble into contigs?
DNA segments prepared from sample as chunks with overlapping regions. As segments are sequenced, computer algorithmically uses the overlaps to assemble the segments into long, contiguous strands called contigs.
List three types of DNA (i.e. not including genes for specific proteins) which would be present in a complete mouse genomic library which would not be present in a cDNA library made from mouse thyroid cells.
(1) Centromere sequences(2) Telomere sequences(3) Introns
Describe how comparative genomics can be used to determine probable protein function.
Comparing gene sequences between organisms and within the genome of one organism to genes of known function and sequence can help suggest a role for the unknown proteins:- Genes with similar sequence (and therefore, similar motifs) in different species are orthologs; similar sequences in same species are paralogs- Presence of either suggests that they have similar functions.Order of genes also can be used to suggest function:- Similar order of genes in one species to genes in another = synteny.Synteny suggests similar function of the similarly order genes in the other species.
Describe GFP and its variants.
Structure: beta barrel with fluorophore in center. Fluorophore made from rearrangement of groups and oxidation of amino acids at center.GFP fluoresces under blue light. Variants have been made to give fluorescence at all different wavelength ranges of visible spectrum.
