Exam 2 Flashcards
How do we name restriction enzymes?
Name derived from the phenomenon
of host-controlled restriction and
modification
What are Type I and Type II restriction
enzymes?
Type I REase: Recognize specific sequences in the DNA but does not cut adjacently to them
* Tracks along the DNA for a variable distance before breaking the DNA strand
Type II REase: Cut within (or immediately adjacent to)
specific target sequences and generate specific fragments
* Only some cut the DNA at a defined distance (only a few bases away) from the recognition site, Limited application
Can we tell the type of restriction enzymes based on the name?
No
What types of ends can be produced from restriction enzymes?
Sticky and blunt
What sequences can be recognized by
Type II restriction enzymes?
Specific short DNA sequences
What are palindromic sequences?
Spelled the same way forward as backward (eg. civic)
What phage groups exist?
tailless, head with tail (e.g., λ), filamentous (e.g., M13)
What are the most common viral vectors?
adeno-associated viral, adenoviral, lentiviral, retroviral
What are the key similarities and
differences between λ and M13 phages?
λ: All regulatory sites are known, not all essential for the phage to function (making it a good place to cut and insert gene of interest)
M13: Does not have non-essential gene, Produce single-strand DNA, Exist as both dsDNA & ssDNA in different phases of its replication cycle
Which phage has ssDNA and which has dsDNA? Are there others that have RNA instead?
M13, yes (MS2 and Qβ)
What is multiplicity of infection (MOI)?
Ratio of “agent” (phase) to “infection target” (cell), Average number of virus particles needed to infect
each cell
How is MOI calculated?
Agent/infection target
What is the MOI if 50 target cells were infected when 1000 viral
vectors were used?
20 = (1000/50)
What are plasmids?
Small, circular pieces of DNA that exist independently of the bacterial chromosome
In what organisms do plamids occur
naturally?
Occur naturally in many strains of bacteria and in a few types of eukaryotic cells, such as yeast
How are plasmids replicated?
Own origin of replication that allows it to be replicated independently of the bacterial chromosome
What types of plasmids exist in nature?
Episome– plasmid
that can integrate
into bacterial
chromosome
What is Conjugation, Transformation and Transduction?
Conjugation: transfer of DNA from a living donor bacterium to a living recipient bacterium by cell-to-cell contact
Transformation: bacterium takes up a piece of DNA floating in its environment
Transduction: A virus transfers chromosomal DNA fragment or a plasmid from one bacterium to another by a bacteriophage
How do we clone DNA into plasmids?
Insert pieces of DNA which are then copied as a part of the plasmid
▫ Pass on to the progeny when the cell replicates
▫ Ability to replicate in the host bacterium
Most, or all, of the enzymes and other products
needed already in the host cell
What are conjugative and non-conjugative plasmids?
Conjugative: Large, low copy # and stringent control of DNA replication (tied to host cell chromosomal DNA replication)
Non: Small, high copy # and
relaxed DNA replication
What are the typical elements of a
plasmid?
Origin of replication, selectable marker, cloning site, reporter genes, promoter, terminator
What are YACs and BACs? What are their advantages and disadvantages?
Used for cloning, can take in big inserts
Yeast artificial chromosomes (YACs): Inserts can be several hundred thousand to 2 million bp long
Bacterial artificial chromosomes (BACs): Inserts can be up to 300,000 bp long
their large insert sizes make them difficult to use in gene cloning and sequencing experiments. Therefore, libraries with smaller insert sizes are needed
How is LacZ gene used for selection?
Blue white screening
What color indicates successful result?
White
How is cI gene used for selection?
If disrupted: undergo lytic
cycle
What indicates success? (cl for selection)
How is an antibiotic resistance gene used for selection?
What do we use transgenic animal cells for?
studying gene function, regulation, and expression. Researchers can introduce specific genes or mutations into animal cells to investigate their roles in cellular processes, disease mechanisms, and developmental pathways.
What is recombination? What are the possible ways?
Cloned genes can be integrated into plant and animal cells through recombination. Gene replacement, Gene addition
What can we achieve with homologous recombination and
non-homologous recombination?
◦ Is one more desirable than another?
Homologous: gene replacement
Non homo: gene addition
Workflow of isolation of nucleic acids
Disruption: lysis of cellular membrane and other cellular components
Separation: precipitation and centrifugation (denser material on bottom)
Recovery: elution of water, separate out
What to use when isolating DNA vs. RNA?
○ When to use RNAse vs. DNAse?
RNA extraction: If an acidic solution of phenol is used, DNA is hydrolyzed and only RNA migrates into the aqueous phase. DNAse added (digest DNA, left with RNA)
DNA extraction: RNAse added (digest RNA), Phenol does not efficiently denature and inhibit RNAse activity
What is PCR?
Polymerase chain reaction (PCR) is the amplification and replication of specific targeted DNA sections targeted by primers
How is PCR similar/different from
natural replication?
targeted amplification to replicate only a segment of DNA bounded by the two primers that determine where DNA polymerase begins replication
What are the common elements in PCR?
Taq polymerase, primers, template DNA, and nucleotides
How does the number of copies
increase over the cycles?
two fold, 2^n
What temperatures are
used for PCR? Why?
Heating up to separate, bring it back down to bind, raise for extension
What chemicals are
needed for PCR?
DNA template, aqueous buffer, buffering agent, master mix, primer, dntp, DNA polymerase
What does ‘RT’ stand for in RT-PCR? What is its purpose?
Reverse transcriptase
produces complementary
DNA (cDNA) from mRNA
What are the two steps of RT-PCR?
Reverse transcription, Amplification
What is real time RT-PCR? What is its purpose?
Real Time rt-PCR (qPCR) provides quantitative results
○ It can identify the starting quantity of copies of the template DNA
○ Fluorescence is proportional to the number of DNA molecules
produced
What is needed for the last step in real
time rt-PCR for TaqMan assay?
How do we quantify expression levels?
The comparative CT method: a widely used method to express relative
gene expression
What is the relevance of the housekeeping gene?
Removes variability from sample size
■ More sample could mean more nucleic acid material, and we don’t
want to mis-interpret this as more gene expression
What are DNA libraries? What are
they for?
collection of DNA fragments
that have been cloned into vectors
▫ so that researchers can identify and isolate the DNA fragments that interest them for further study.
What main types of DNA libraries
exist? What are the key differences?
genomic: set of clones representing the entire genome of an organism, and the production of such a library is usually the first step in isolating a DNA sequence from an organism’s genome. contain large fragments of DNA in either bacteriophages or bacterial or P1-derived artificial chromosomes (BACs and. PACs).
cDNA: made with cloned, reverse-transcribed mRNA, and therefore lack DNA sequences corresponding to genomic regions that are not expressed, no introns and 5′ and 3′ noncoding regions of eukaryotic
gene. Advantage for making protein from eukaryotic gene in prokaryotic cell because they can use it without the introns, but they cannot use it with the
introns. contain much smaller
fragments than genomic DNA libraries, and are usually cloned into plasmid vectors
How are DNA libraries produced?
(steps: isolation, digestion, insertion,
amplification)
extract and purify DNA
Digest DNA with restrictor enzymes
Insert into plasmids
How can we retrieve a clone that
contains a specific sequence?
Primers, PCR
Which direction is DNA moving in
the gel? Why?
Down towards the positive, DNA is negatively charged.
Why does smaller DNA move
faster?
It is lighter, less nucleotides
Is this the sequence of the
original strand? If not, what is the
original sequence? (sanger)
No, it is the strand complementary to the sanger.
What are we detecting
specifically with western blots?
Proteins
What are the probes in the case
of Western Blot?
Antibody based probes
What is the purpose of these
blotting techniques? (northern and southern)
Northern: detect RNA
Southern: detect DNA
What is the difference between
Southern and Northern blot?
Northern detects RNA, Southern detects DNA
What are the steps? What is the
purpose of the membrane?
Why is it placed on the top of
the gel? (northern and southern)
The membrane serves as a solid support onto which the nucleic acids from the gel can be transferred, allowing for easier handling and analysis. Placing the membrane on top of the gel ensures efficient transfer of nucleic acids by capillary action, as the gel and membrane are in close contact during the transfer process.
What are the probes? (blotting)
Northern and southern: Oligonucleotide Probes
What do you understand about the meaning of different values of P-value, Log2ratio, etc when looking at gene
expression data?
The Log2ratio, also known as fold change, represents the magnitude of change in gene expression levels between experimental conditions
How does bioinformatics help us? (experiment design, data analysis, databases)
Interface between computer science and molecular biology. It involves the use of computers to store, organize, and interpret biological information, usually in the form of sequence data.
How do we handle data from sources with different naming conventions?
Standardization, cross-referencing
What is the GIGO effect? Why is it important?
stress the importance of valid and reliable data entry when programming or entering information. interrogation of databases can provide some novel insights
What is/are required for creating
recombinant DNA in the “cut and paste/join” process?
Restriction enzymes and DNA ligase
What type of restriction enzyme is
commonly used in GE?
Endonuclease (cut in specific places, cut nucleic acid molecules internally but not degrade from the ends of nucleic acids)
What does ‘restriction enzyme’ break
directly in DNA?
Phosphodiester
bond between
nucleotides on the
same DNA strand
Which type(s) of restriction enzyme
is/are useful for GE when accurate cut
at specific places is required?
Type II
Chain-terminating dideoxynucleotides (ddNTP’s).
* ddGTP, ddATP, ddTTP, ddCTP
nearly identical to nucleotides except for the fact that they are fluorescent and have an -H instead of -OH on the 3’ carbon
