Hybridization

increase the salt concentration can create an _____ which creates a false positive

artificial hydrogen binding

why will SyBrGreen I not mark the DNA past the melting temperature

because after dsDNA gets to the melting temperature it becomes single stranded so SyBrGreen can not longer mark it

why do we use non-denaturing gel electrophoresis in SSCP

because we are concerned with shape and not size

if conditions of stringency are set too low, then the probe will ____

bind unrelated targets and thus result in false data

the main commonality between SSCP and heteroduplex is

both assays detect the formation of partial duplex DNA that could be separated by a non-denaturing capillary gel electrophoresis

the target is _____ to the probe

complementary

the two system that capillary electrophoresis can be run

completely denatured and non-denatured condition

can design a positive and negative ____ to set stringency

control

after we heat to DNA so its becomes denatured, we slowly ____

cool

in heteroduplex analysis, we heat to ____

denature dsDNA

heteroduplex analysis involves _____ and ____

denaturing and hybridization

the target is the nucleic acid we want to ____

detect

in the condition that ____ or in the _____ of complementary strand, the ___ ssDNA forms intra-strand duplexes

disfavors; absence of complementary strand; diluted

nucleic acid hybridization is the formation of ______ between two ______ via a sequence specific interaction

double stranded duplex; complementary single strand molecules

SyBrGreen I marks ____

dsDNA

at melting temperature, half of the sequence is ______ while the other half is _____

dsDNA; ssDNA

when the target and probe come together, they form a ____ molecule

duplex

if condition of stringency are set too low, then the probe will bind unrelated targets and thus result in ____

false data

the dissociation of dsDNA to ssDNA as temperature rises can be monitored by the use of ____

fluorescent dye that binds only to the dsDNA

usually a ____ label is applied to the probe as an enzyme tag

fluorescent label or radioactive enzyme

lower stringency conditions are rather ____

forgiving

occasionally, if denaturant like ____ is included in the buffer, then its concentration could so affect stringency

formamide

why would we not want to add formamide, urea, and heat to the non-denaturing gel electrophoresis of SSCP

formamide, urea, and heat would denature the DNA

clinical molecular labs attempt to visualize and detect a particular ____ or ____ of interest

gene; region

____ analysis is the mixing of nucleic acid fragments with reference fragments followed by denaturing and hybridization that can detect mutations

heteroduplex analysis

the higher the stringency, the ___ perfect correctly matched hybridization between the probe and the target nucleic acid in specimens

higher

term meaning the combination of conditions under which the target is exposed to the probe

hybridization stringency

lowered temperature or elevated salt concentration can result in a forgiving condition and thus permit the formation of ____

incorrect heteroduplexes

in the condition that disfavors or in the absence of complementary strand, the diluted ssDNA forms _____

intra-strand duplexes

the target is usually ____ than the probe

larger

capillary electrophoresis can be run in a completely denatured condition to determine the ____

length of DNA fragments carrying similar sizes with a few bases in difference

____ and the ____ of the probe sequence could affect stringency

length; nature of the probe

after the hybridization assay, if negative signal was obtained in the "positive control" probe, the improvement strategy would be to

lower hybridization temperature raise hybridization salt concentration

the probe is commonly ____

marked or labeled

____ exploits the sequence and stacking directed denaturation characteristics of DNA duplex

melt curve analysis

assay the requires the use of fluorescent dye for binding ssDNA

melting curve

involves data generated from both assays is indicative of GC%

melting curve

the main commonality between melting curve analysis and single-strand conformation polymorphism is _____

melting of dsDNA into ssDNA; ssDNA then becomes an important target to be detected in both assays

_____ is the opposite of hybridization

melting temperature

the ideal hybridization conditions are inferred form calculation of the ____

melting temperature of the given probe sequence

at Tm, because half of the DNA sequence is double stranded and the other half is single stranded, Tm is the ____ of the melting curve

midpoint

a long probe or one with higher GC will bind under ____ stringent conditions, than a short probe

more

heteroduplex analysis allows you to detect ____

mutations

_____ is the formation of double stranded duplex between two complementary single strand molecules via a sequence specific interaction

nucleic acid hybridization

the amount of intra-strand duplexes is mainly determined by ____

nucleic acid sequence

the migration of single stranded conformer in _____ or in ____ under precisely controlled non-denaturing _____ conditions distinguishes sequence of variants even through single-base mutations

polyacrylamide; gel electrophoresis; temperature

the proper stringency is guided by how specimen hybridized to ___ probes

positive and negative probes

the 3D structure and shape of the intra-strand duplex is determined by the _____

primary sequence of the folded strand

____ is a single stranded sequence of nucleic acid that is complementary to the nucleic acid sequence of interest

probe

either the probe or the target DNA can be labeled, but generally ____ is labeled

probe

the first things we need to do is to denature dsDNA so that the _____ can bind to ssDNA

probe

the test reagent in nucleic acid hybridization

probe

the condition of high stringency are more demanding of the ____

probe/target complementarity and length

after the hybridization assay, if a positive signal was obtained from the "negative control" probe the improvement strategy would be to

raise hybridization temperature and lower hybridization salt concentration

high stringency has ____ mismatch

rare

the reference fragments are in the _____

reagents

the non-denaturing gel electrophoresis used in SSCP allows you to visualize ____

secondary structure

the migration of single-stranded conformer in polyacrylamide or in capillary gels electrophoresis under precisely controlled non-denatureing and temperature conditions distinguishes _____

sequence of variants even though single-base mutations

capillary electrophoresis can be run in a non-denatured condition to assess the subtle differences in ____

shape or confirmation of the target nucleic acids

the probe is _____ sequence of nucleic acid that is complementary to the nucleic acid sequence of interest

single stranded

_____ is based on preference of ssDNA to exist in a partially dsDNA rather than completely single stranded state

single-strand conformation polymorphism (SSCP)

capillary electrophoresis ran in non-denatured condition disregards ____

size

the probe is generally a _____

small strand of ssDNA

the nucleic acid fragments are in the ____

specimens

SyBrGreen II marks ____

ssDNA or ssRNA

single strand conformation polymorphism is based on the preference of ____ to exist in a ____ rather than completely single stranded state

ssDNA to exist in a partially dsDNA

GelRed marks ____

ssDNA/ssRNA and dsDNA/dsRNA

SyBrGold marks ____

ssDNA/ssRNA and dsDNA/dsRNA

____ is the nucleic acid of interest

target

you can increase stringency by increasing _____, lowering ____, and increasing _____

temperature; salt concentration; increase formamide concentration

melting temperature is a way of correlating the amount of ______ required to _____

temperature; separate hybridized strand of a given sequence

high stringency give you ____ base pairing and ____ mismatch

true; low

capillary electrophoresis can be run in ___ systems

two

heteroduplexes can be detected by ____ methods

two

heteroduplexes are formed when _____ sequences occur

unmatched

if conditions of stringency are set too high, then the probe ____

will not bind to its target

heteroduplexes can be detected by what two methods

1. formation of heteroduplex slows down migration 2. the un-matched region of the heteroduplex can be cleaved by S1-nucleases and thus results in fragments

process of heteroduplex analysis

1. get the reference and specimen DNA 2. heat and then cool to 25oC 3. cool to denaturing temperature 4. hybridization

steps of SSCP

1. take DNA specimen with reagent and denature to make ssDNA 2. heat to melting temperature to make ssDNA 3. heating to melting temperature creates secondary structure due to intra-molecule base pairing 4. analyze on non-denaturing gel

the main factors affecting stringency include _____

1. temperature at hybridization 2. salt concentration of the hybridization buffer

optimal hybridization temperature is determined by ____

1. types of nucleic acid hybrids 2. probe sequence

how many base pairs are normally in a probe

20 base pairs

the phenomenon of intra-strand duplexes results in a ____

3D structure and shape

the hybridization temperature of oligonucleotide probes can be tentatively set at ___ below the melting temperature

5oC

the hybridization temperature has to be ___

5oC below Tm

what would we want our starting stringency temperature to be

5oC below the melting temperature

the higher the concentration of _____ the higher the melting point

GC

why would higher concentration of GC cause melting point to rise

GC are connected by a triple hydrogen bond

probe sequence refers to ____

GC% content

normally, ____ hybrids will require a higher temperature than ____ hybrids

RNA:RNA > RNA:DNA > DNA:DNA

an extra fragment will derive from heteroduplexes after ____ digestion

S1-nuclease

assay that requires non-denaturing gel electrophoresis

SSCP

involves detecting the ability to form intra-stand duplexes

SSCP

how is SSCP the opposite of capillary electrophoresis

SSCP is concerned with shape; capillary electrophoresis is concerned with molecules of similar size

____ will mark dsDNA in the Tm curve

SyBrGreen I