ANSC 4050 Test 1
where is the start of transcription
+1 and this is downstream
what characteristics should a primer have when you design it
18-30 bp; 40-60% G-C content; TM of 55-75 C
how many hydrogen bonds does A-T have
2
how many promoters does RNA polymerase II bind
2; TATA box and CAAT box
what technology is used in proteomics
2D gel electrophoresis to separate the proteins by charge and size and then you do Mass Spectrometry to identify proteins after separation and then you use bio informatics to link results
how many hydrogen bonds does G-C have
3
how long are bacterial (prokaryotic) promoters
30 nucleotides
what temperature do the primers anneal
30-65 C
what are 3 majors forms of processing during transcription
5' cap; poly A tail; splicing
how is DNA read
5'-3'
what breaks the probe apart in taqman to free the reporter
5'-3' exonuclease activity
how long are enhancers
50-100 bp ; can be upstream or downstream
what is the initiator codon
AUG --codes for methionine
what is the most widely used vector for genome sequencing projects
BACs
how are DNA libraries made
DNA from extracted tissue and a separate vector are both treated with restriction enzymes; then the clonable DNA is put into the vector and the ligated vector/target DNA is introduced into bacteria
how can libraries be screened for certain components
DNA hybridization and immunological assay
replication is carried out by which enzyme
DNA polymerase
what is elongation
DNA unwinds and ribonucleotides are added to 3' end of newly synthesized RNA molecule
what is the central dogma
DNA-->RNA-->protein
transcription is carried out by what enzyme
RNA polymerase
what unwinds the DNA to start transcription
RNA polymerase and transcription factors
what is fast becoming the method of choice for gene expression analysis
RNA-sequencing
what are 2 examples of artificial chromosomes
YACs and BACs
what is a proteome
a catalog of all the proteins throughout the life of the organisms and under all conditions
what is first generation DNA sequencing
a completed cyclic reaction and an analysis of terminated fragments at the end of the reaction
what does a trigylcercide consist of
a glycerol backbone and 3 fatty acids
what are 4 things that are required for replication to occur
a template DNA, helices (to unwind DNA), DNA polymerase, DNA ligase
what are the key advantages of ddPCR
absolute quantification; can detect rare targets
what are reasons TO DO taqman
allows you to do multiplexing
what are proteins made up of
amino acids
what is the product of a PCR called
amplicon
what is the Sanger method
an in vitro DNA synthesis method that involves a DNA primer, labeled nucleotides, ddNTPs, and 4 dNTPs
what is biotechnology
any technique that uses living organisms or substances from those organisms to make or modify a product, to improve plants or animals, or to develop microorganisms for specific uses
how many resection enzymes are known
around 3,000; 600 are available commercially
how many oligonulceotides can you on a chip
as many as 350,000 but its limited by sequence info or verified cDNA clones
where are the location of the promoters
at 10 and 35 nucleotides upstream
where does the majority of gene expression occur
at the transcriptional control area (promoters and enhancers)
why has capillary electrophoresis become so important in molecular biology techniques
automation
what does first dimension 2D electrophoresis separate on
based on charge
where does RNA polymerase bind
binds to a promoter in upstream region near the start of transcription
what is similar about replication in prokaryotes and eukaryotes
both replications are bidirectional
what are palindromic sequences
both strands are the same forwards and backwards
what is an example of a splice variant
calcitonin --one variant increases calcium uptake and the other variant causes blood vessels to dilate
what is genomics useful for studying
cancer and hereditary metabolic disorders
what are polysaccharides made up of
carbohydrates
what is a protein
chain of amino acids connected by peptide bonds
what are 4 ways to affect the rate of migration
changing strength of electrical field, changing ionic strength of the buffer, change the temperature, based on size
what is a charged amino acid
charged at physiological pH which means if there is change in the pH these charged amino acids will lose or gain a charge
what are the 3 most common ways to classify an amino acid
charged, polar, non polar
where does photosynthesis occur
chloroplast
what are plasmids
circular double stranded DNA that carries antibiotic resistance and are small so they are easily isolated
what shape is the DNA in prokaryotes
circulate
what is the cDNA gene array protocol
collect RNA then make cDNA from the RNA via reverse transcriptase and a label will be incorporated in some way and then mix them all together and put them on the array
what are -omics
collection of analytic tools that attempt to look at the entire population of something all at once
what does reverse transcriptase do
copies RNA into DNA
what is cDNA
copy of mRNA
what is synthetic biology
creating new biological parts, devices, and systems
what is the purpose of the poly A tail
cuts the transcript to a specific length to stabilize it
where does replication occur in prokaryotes
cytoplasm
where does transcription occur in prokaryotes
cytoplasm
where does translation occur in eukaryotes
cytoplasm
where does translation occur in prokaryotes
cytoplasm
what is the function of restriction enzymes
degrade foreign DNA (viruses)
what is an amplicon
double stranded DNA that is a defined length and is a sequence between two primers and the ends are defined by primer sequences
what are the spots on micro arrays
each spot represents a different DNA sequence
what does the concentration of agarose effect
effects the size of the pores--the more agarose, the smaller the pores
how does southern blot separation occur
electrophoresis
what are the steps involved in blotting techniques
electrophoresis; blotting or transfer; and then probing
where does the synthesis of lipids occur
endoplasmic reticulum
where does the post translational modification of a protein occur
endoplasmic reticulum or golgi
the cell specificity of transcription is enhanced by what
enhancers
what are different methods to visualize in the agarose gel
ethidium bromide nucleic acid strain; SYBR green
what is the difference between prokaryotes and eukaryotes
eukaryotes have membrane bound organelles
what are reasons to not do Taqman
expensive and lots of quality control compared to SYBR green
what does a cDNA library represent
expressed genes of particular tissue
what are lipids made up of
fatty acids
how is drop digital PCR read
flow cytometer
what is the goal of digital PCR
for each partition to represent one molecule of template
what happens when agarose is cooled
forms double helix regions that create the pores
what are micro arrays used for
gene expression analysis; a segment of DNA representing a particular gene is spotted in a certain spot on the array so that the spot represents if that gene is being expressed in a cell type or tissue
what is differential expression
gene is more highly expressed in one treatment than in another treatment its compared to
what codes for proteins
genes
what is the structure of a phospholipid
glycerol backbone, 2 fatty acid chains, and a phosphate group that is linked to another group
What is RNA sequencing?
going from mRNA to cDNA and making gene transcripts
where does lipid secretion occur
golgi apparatus
what 4 things are required for a PCR
heat (96 C); heat stable DNA polymerase; primers flanking area; thermocycler
what purpose does 5' capping have
helps with transport, protection, and involved in translation
what constitutes a good DNA library
high representation; high proportion of original fragment is represented
how does southern blot detection occur
hybridization on membrane
what are cosmids
hybrids of phage DNA nad plasmids
what holds the 2 strands of DNA together
hydrogen bonds
what is the polar nature of phospholipids
inherently polar; one end is not the same as the other end
what is Ct (cycle threshold analysis)
instead of measuring the change inflorescence at a set cycle number, you measure the number of cycles it takes to reach a set fluorescence threshold
what is spliced out during splicing
introns
what is the western blot
is used identify specific proteins
what is the northern blot
is used to identify specific mRNA sequences
what does Taq Polymerase do
it attaches nucleotides to the DNA ; also has proofreading abilities
what is the restriction of first generation DNA sequencing
it can only be run one sequence per reaction
what is isothermal amplification and what uses it
it does not require changes in temperature; you only need one temperature the whole entire time; the RPA cycle uses it
what does the 5'-3' exonuclease do in Taqman
it extends the DNA polymerase in that direction and if it comes across double stranded segment it'll remove it
what are fatty acids made up of
it is a carbon back bone; if there are no double bonds it is saturated, if there is 1 double bond it is unsaturated
why do we use taq polymerase
it is heat stable so it is stable in the elevated temperatures
what happens to agarose as it is heated
it is in loose random chains
what causes DNA to migrate towards anode
its charge because the anode is positive and DNA is negative
what is a DNA library
large collection of DNA fragments, cDNA, or fragments of genomic DNA
what are macromolecules
large molecules made up of smaller units
what is the upside of agarose
large range of separation (200-50,000 bp)
what shape is the DNA in eukaryotes
linear
what is the downside to agarose
low resolving power
what are 2 parts of translational control
mRNA stability and miRNA
what are the main advantages of digital PCR
makes single cell analysis possible
what is agarose
matrix that is a linear polysaccharides that can have a 0.5-2% concentration
what are the 3 major steps in a PCR reaction
melting; annealing of primers; and elongation or extension
what is the southern blot
method for separating and identifying specific DNA oligonucleotide sequences
what are 2 ways to do a digital PCR
micro fluidic or drop digital
where does production of ATP occur
mitochondria
what does second dimension 2D electrophoresis separate on
molecular size
how many origins of replication are in eukaryotes
multiple origins
why is PCR DNA Sequencing better than the Sanger Method
multiple rounds of synthesis occur and there are fewer copies of the target sequence so it is faster and P32 can be replaced with fluorescent labeled ddNTPs so that all systems fluoresce with different colors
how are restriction enzymes named
named after bacteria in which it was discovered; first letter is capitalized and is from genus; second and third letters are the first 2 letters of the species; then has certain letters to represent the strain and if there is a roman numeral it indicates order it was discovered
what type of charge does DNA have
negative
do only eukaryotes have ribosomes
no, they are not membrane bound organelles
is biotechnology a discipline?
no; it is an application of disciplines such as biochemistry, cell biology, and engineering
what are the 3 properties of restriction endonuclesuease described by the name
nuclease; cuts within the sequence; cuts at a specific site
what are nucleic acids made up of
nucleotide bases
where does DNA replication occur
nucleus
where does replication occur in eukaryotes
nucleus
where does the transcription of a gene occur
nucleus
where does transcription occur in eukaryotes
nucleus
what are affymetrix gene chips
oligonucleotide are synthesized on silicon chips; the oligonucleotide for each gene are unique in the genome and are non overlapping
how is DNA represented on micro array
oligonucleotides
translation is performed where
on ribosomes
how do you analyze microarray results
one condition is compared to another; analysis algorithms can be used and there is inherent variability
how many origins of replication are in prokaryotes
one origin
what are 3 things required for cloning vectors
origin of replication; unique insertion site, some means of selection
if an amino acid is normally charged, what might charge to eliminate the charge
pH
what links the 3' carbon to the 5' carbon in DNA
phosphodiester links
what are 4 ways proteins can be post translationally modified
phosphorylation, cleavage, adding cofactors, sulfation
what is the most common cloning vector
plasmids
what is hyaluronan
polymer of disaccharides that is the major component of extracellular matrix and cartilage
what components of the PCR reaction are incorporated into the PCR product
primers and dNTP's
what is added to the PCR reaction
primers, dNTPs, Mg, buffer, Taq polymerase
what is Taqman
probe based detection; this is when a probe is added to the PCR reaction
how does Taqman work
probe contains a reporter and a quencher and the reporter cannot fluoresce when the quencher is also on the probe but if the reporter is released it can fluoresce
what is a transgene made of
promoter + gene
what are modification enzymes
protects the bacterial DNA by adding methyl groups to the restriction site
what are transcription factors
protein that controls the rate of transcription
is real time PCR qualitative or quantitative
quantitative
how is transcriptomics usually preformed
quantitative real time PCR
How is translation initiated?
rRNA and initiator tRNA bind to mRNA
In the Sanger Method, what is the labeled nucleotide in the newly synthesized DNA
radioactive phosphate (P32)
what are the 3 enzymes in the RPA cycle
recombinase; polymerase; and single strand binding protein
how do cosmids replicate
replicate as plasmids and do not lyse cells
what enzyme is used to synthesize cDNA
reverse transcriptase
where does the translation of an mRNA into a protein happen
ribosome
what is capillary electrophoresis
sample moves through capillary tubing under electrical field; separates on the basis of charge and size
what does reading the sequence in real time mean
sequence is read at each position sequentially rather than at the completion of reaction
what are restriction fragments
short strands that are left after the enzymes cleave both strands of DNA
what is so good about next generation DNA sequencing
shorter read lengths, cheaper, makes the idea of personal genome sequencing practice
how does mRNA become cDNA
single strand of mRNA becomes a single strand of cDNA and you use the PCR technique to make a second strand so now you have double stranded cDNA
what is the main reason for using something other than a plasmid as a cloning vector
size of insert is too large
do we separate with shape or size
size; smaller molecules will move faster
what are DNA microarrays
slide spotted with either PCR products
what are splice variants
splicing isn't always does the same way everytime so this leads to the same genes producing multiple proteins
how is translation stopped
stop codons; UAA, UAG, UGA
what is lock nucleic acid
synthesized nucleic acid link between the adjacent ribose molecules that lock them so they can't rotate
what are YACs and BACs
synthetic cloning vectors which replicate in host cells like extra chromosomes
what is a digital PCR
taking a normal PCR reaction and dividing it up into a bunch of little reactions and you partition it into separate reactions
what are restriction sites
the 4-8 bp sites that restriction enzymes recognize
what is the melting temperature of a primer determined by
the GC content--because the more GC you have the higher the melting temp needs to be
what is the primary structure for proteins
the amino acid sequence
what determines the separation range in regards to agarose
the amount of agarose
in regards to real time PCR--what should the amount of amplification product be relative to
the amount of amplification product needs to be proportional to the amount of template you start with
what does it mean that the PCR reaction saturates
the amount of product is not proportional to the amount of template; this means you have to look at it in the early cycle of the PCR reaction
what is proteomics
the analysis of an organisms' proteome
what are the 2 different ends of a phospholipid
the end with 2 fatty acids is hydrophobic; the other end is hydrophilic
A large double stranded DNA molecule is completely digested with the Restriction Endonuclease EcoR I. What unequivocal statement can be made about the fragments generated?
the ends of the fragments are the same and defined by the enzyme used
what is the secondary structure for proteins
the flooding of the amino acids
what are the post translational modifications that occur
the folding of the end over on itself, and then most Met is removed from the end
what is the tertiary structure for proteins
the interactions between the folded structures
what is transcriptomics
the measure of gene expression and of which genes are turned on/off
what is electrophoresis
the separation of molecules with an electric charge
what are challenges within proteomics
the splice variants cause lots of diversity and post translational modifications can change protein function
what is genomics
the study of total genes in an organisms
what is the purpose for phospholipids
they are the main component of membranes and form a lipid bilayer
what is a codon
three nucleotide code for insertion of an amino acid
what is the purpose of a PCR
to amplify selected regions of DNA
what is the goal of proteomics
to catalog all proteins, to understand their functions, and ot understand how they interact
what is the southern blot used for
to characterize cloned DNA, to map restriction sites in or near a gene; to identify specific DNA fragment from a mixture of fragments; to identify related genes in different species; to detect rearrangements or duplication of genes in genetic disorders
what are YAC and BAC used for
to clone very large segments of DNA (100-300 kb)
what can proteomics be used for
to compare different cell types; to look at the cell or tissue response to certain conditions; or to compare cancer and other disease profiles
why is SYBR green used
to detect the accumulation of PCR product
what makes up the master mix
tris buffer; primers; MgCl2; dNTP; dH2O; taq polymerase
what is complimentary base pairing
two strands of DNA come together and two nucleotide bases are paired with one another; G-C and A-T
what happens in polyacrylamide denaturing gel
urea converts double to string strand; uses SDS for proteins
what is polyacrylamide (PAGE)
used in sequencing gels; gives good resolution; 5-2,000 bp; oxygen inhibits polymerization so it must be poured between glass plates
what is a bacteriophage
virus that infects bacteria
what is absolute quantification
when there is absolute count of copies of the target; no standard curve or relative quantification
when is recombinant DNA (rDNA) formed
when two or more pieces of DNA are put together to form a new DNA molecule
how do you monitor the accumulation of the product in real time
you can compare to other samples or compare between samples or against standards
how does DNA hybridization work
you match a sequence with a labeled complementary sequence and then this is transferred to nitrocellulose and you look for the label
what is drop digital PCR
you partition PCR samples into water-in-oil droplets and there is PCR amplification in each drop and each drop is analyzed or read by flow cytometer
how does immunological assay work
you transcribe and translate DNA into a protein and then use an antibody to protein look for Ab-Ag binding
how is cDNA amplified via PCR
you use sequence specific primers
