methods of genotyping Flashcards
DNA polymorphisms
sequence difference compared to a reference standard that is present in at least 1% of a population
can be single bases or thousands
may or may not have phenotypic effects
found througout the genome
each polymorphic marker has different versions or alleles
they can change the nucleotide sequence or lenght
genetic polymorphism analysis
4 types of polymorphisms commonly used for analysis
- RFLP
- VNTR
- STR
- SNP
key terms
alleles- alternative forms of DNA sequence/fragments (2 alleles per person at a locus)
homozygotes- same type of of alleles (cc or 3-3 repeats)
heterozygotes- different types of alleles (CT or 2-3 repeats)
single nucleotide- one nucleotide changes
length polymorphisms- changes the length of the sequence an extra repeat
single nucleotide polymorphisms (SNPs)
purines- A G
pyrimidines- C T
purine with purine or pyrmidine with pyrimidine= transitions
A with C or T= transversions
transitions are more common than tranversions causing less instability in the DNA helix
synonymous substituions (silent) are less likely to impact phenotypes than non- synonymous subs (replacement) as they do not change the protein sequence
common dna polymorphisms
dna polym are analysed by changes in the nucleotide sequence or size
length polymorphisms
RFLPs (restriction fragment length polymorphisms) - variable base pairs- original method of SNP analysis
VNTRs (variable number of tandem repeats) - large size polym - 10-100bp- require high quality, rarely analysed in normal research
southern and northern blotting
polymorphism detection methodology
purpose- make a permanent record of results of gel electropheresis
souther blots= DNA
northern blots= RNA
western blots= proteins
eastern blots= PTM (post translational mods)
southern blotting
fragmented dna is separated by electropheresis
dna fragments are denature wirh alkali + transferred to a nitrocellulose membrane by capillary action- creating a replica of the original gel
membrane is incubated with labelled probe which binds to and detects the fragment of interest
need to know the seqeunce we are dealing with to get the probe to match
southern blotting steps
- extraction and purification of dna from cells - dna separated from target cells
- restriction digestion, dna fragmentation or differential pcr - RE cut high MW dna strands into smaller fragments
- separation by electropheresis - distance moved depending on size
- chemical modification - dna is denatured with mild alkali such as NaOH
- transfer/blotting- denatured fragments transferred onto a nylon or nitrocellulose filter membrane
- hybridisation- membrane exposed to hybridisation probe - probe dna is labelled sp that is can be detected
- washing of unbound probes
- detection- hybridised regions autoradiographically by placimg nitrocellulose membrane in contact with photographic film which shows hybridised dna molecules
northern blotting
detects the prescence of particular mRNA in a sample
transfer RNA from electrophoretic gel to modified nylon membrane
nucleic acid probe hybridisation
single stranded dna probe and a single stranded target rna
doesnt require denaturation as rna is single strnaded- requires destruction of any secondary structure formed by rna
restriction enzymes
restriction fragment length polymorphisms (RFLPs)
cut out dna at specific sequences
restriction fragment sizes aew altered by changes in or between enzyme recognition sites
if there has been mutation, the restriction enzyme will cut ina different way, producing new fragments of different lengths which can be used to identify any polymorphsims
dna polym are analysed by changes in the nucleotide sequence or length
pcr-rflp
reactions designed to produce products of different sizes after RE cleavage
pcr with specific primers to target the region of interest and then digestion with appropriate/ different REs
fragment can then be identified on gel
RFLP genotypes are inherited for each locus, one allele is inherited from each parent- can be used to study hereditary patterns
pcr rflp adv + dis
adv
- simple
- quick digestion
- inexpensive
- effective
dis
- low sensitivity (high conc of sample needed)
- prior knowledge of dna sequence needed
- non automated
- multiple steps
- labour intensice
short tandem repeat polymorphisms
STR are repeats of nucleotide sequences
interested in the number of times a sequence is repeated- not what a sequence is
STR repeats can be analysed by fragment size (southern blot)
STR alleles analysed by amplicon size (pcr) can be detected by fluorescence if flurophore attached to primers
dna profile
unique regions of human genome are targeted
regions consist of a few hundred base pairs
regions are copied by PCR- billions of exact copies made
copied fragments now comtain fluorescent dyes for detection
STR categories
simple repeats- contain units of identical length + sequence
simple repeats with non consensus alleles
compound repeats- comprises of 2 or more adjacent simple repeats
complex repeats- contain several repeat blocks of variable unit lengths
(table of exmample in notes)
advantages for STR markers
small product sizes are generally compatible with degraded dna and pcr enables recovery of information from small amounts of material
multiplex amplification with fluorescence detection enables high power of discrimination in a single test
commerically available in an easy to use kit format
uniform set of core STR loci provide capability for national and international criminal dna profiles
ALFP-PCR
amplified fragment length polymorphism
uses selective amplification of a section of digested dna fragments to generate unique fingerprints for genomes of interest
can quickly generate large numbers of marker fragments for any organism- without prior knowledge of sequences
RE digest genomic DNA and allow attachment of adaptors to sticky ends
part of restriction fragments is selected to be amplified by using primers that are comp to adaptor sequence
amplified sequences are separated and visualised on agarose gel electropheresis
RAPD
random amplification of polymorphic dna
-amplified dna is random- no knowledge of dna sequence in targeted dna
short primers are added into dna and see what is amplified
genomic dna
electrophoretic banding pattern- semi unique profile
amplification will only take place in regions of the genome that has the sequence complementary to the deocoligonucleotide at both ends
if there has been mutation in template dna (in site that was previosuly complementary) PCR product will not be produced= different pattern of dna on gel
used in identification and quantification of pathogens, assessment of genetic diversity
quantitative pcr - qPCR
based on detection and quantification of a fluorescent reporter
monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each pcr cycle in real time (as apposed to endpoint detection)
concentration of the nucleic acid present into the sample is quantified using
fluroscent dye
can use 2 fluorogenic probes to discriminate between 2 different alleles
q-pcr is applied in genotyping and quantification of pathogens, microRNA analysis, cancer detection, microbial load testing and GMOs detection
ARMS
amplification refractory mutation system
allele specific pcr
exploits the differential efficiency of pcr primer extension when it is eiteher matched or mismatched to a template at its 3’ end
ARMS primers act as PCR switches either inhibiting or enabling PCR - depending on whether the primer matches the allele on the template being amplified
ARMS primer specificity is a measure of the relative priming efficiency on a matched versus mismatched template
used in selective quantification of dna, medical and diagnositc approaches, forensic studies and research areas
conventional (end point) pcr
allows a target dna sequence to be selectively amplified several nmillion folds in a few hours
enables synthesis of specific dna fragments using dna poly
dna poly synthesises comp sequence of dna as a primer is connected to one of the dna strands in the specific site
primers limit the sequence to be replicated + results in the amplification of a specified dna sequence with billions of copies
applies in selective dna isolation, amplification + quantification of DNA, medical and diagnostic approaches, infesctious disease studies, forensic studies + research
digital pcr
quantitative pcr technology that measures amount of dna or rna present in a sample
initial sample is divided into wells
means that target seqeunce is either in well or not in a well
based on presence or absence of fluorescence in the amplified reaction wells - can calculate the absolute number of targets present in the original sample
wells with fluorescence are positive= 1
wells with no signal are negative= 0
conc of target sequence present in initial sample is determined through poisson statistical anaylsis
dpcr is used to determine total number of dna and rna viruses, bacteria and parasites in a variety of clinical specimens
limitations of end point pcr
poor precision
low sensitivity
short dynamic range
low resolution
non automated
post pcr processing
real time pcr adv + dis
adv
- traditional pcr is measured at end point whereas real time collects data in exponential expansion phase
- increased dynamic range of detection
- no post-pcr processing
dis
- due to high sensitivity, more error prone
- requires expensive equipment + reagents
- requires high technical skill + support
melt cureve on mRNA analysis
high resolution melting
- melting curve charts the change in fluorescence observed when dsDNA with incorporated dye molecules melts into the ssdna as the temp of the reaction is raised
- heating double stranded DNA bound with sybr green 1 dye results in a decrease in fluorescence when melting point is reached
- fluorescence is plotted againts temp and change in flu/temp is plotted against temp to obtain clear view of melting dynamics
adv of digital pcr
enhanced sensitivity
precise quantification
mitigates target competitiion
pcr amplification less sensitive to inhbition
enhanced discriminatory capacity of assays that differ by only a single nucleotide
rare sequence detection
doesnt rely on the no. of amplification cycles to determine initial amount of template nucleic acid
