1 RFLP analysis RFLP= Restriction fragment length polymorphism Refers to variation in restriction sites between individuals in a population These are extremely useful and valuable for geneticists (and lawyers) On average two individuals (humans) vary at 1 in 1000 bp The human genome is 3x10 9 bp This means that they will differ in more than 3 million bp. By chance these changes will create or destroy the recognition sites for Restriction enzymes
RFLP analysis. RFLP= Restriction fragment length polymorphism Refers to variation in restriction sites between individuals in a population These are extremely useful and valuable for geneticists (and lawyers) On average two individuals (humans) vary at 1 in 1000 bp - PowerPoint PPT Presentation
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RFLP analysis
RFLP= Restriction fragment length polymorphismRefers to variation in restriction sites between individuals in a populationThese are extremely useful and valuable for geneticists (and lawyers)
On average two individuals (humans) vary at 1 in 1000 bpThe human genome is 3x109 bpThis means that they will differ in more than 3 million bp.
By chance these changes will create or destroy the recognition sites for Restriction enzymes
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RFLP
Lets generate a restriction map for a region of human X-chromosome
The restriction map in the same region of the X chromosome of a second individual may appear as
Normal GAATTC
Mutant GAGTTC
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RFLP
The internal EcoRI site is missing in the second individual
For X1 the sequence at this site is GAATTCCTTAAG
This is the sequence recognized by EcoRI
The equivalent site in the X2 individual is mutated
GAGTTCCTCAAG
Now if we examine a large number of humans at this site we may find that 25% possess the EcoRI site and 75% lack this site.
We can say that a restriction fragment length polymorphism exits in this region
These polymorphisms usually do not have any phenotypic consequences Silent mutations that do not alter the protein sequence because of redundancy in Codon usage, localization to introns or non-genic regions or do not affect protein Structure/function.
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RFLP
RFLP are identified by southern blotsIn the region of the human X chromosome, two forms of the X-chromosome are Segregating in the population.
Digest DNA with EcoRI and probe with probe1What do we get?
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RFLP
Digesting with BamHI and performing Southern blots with the above probe produces the following results for X1/X1, X1/X2 and X2/X2 individuals:
There is no variation with respect to the BamHI sites, all individuals produce the same banding patterns on Southern blots
If we used probe2 for southern blots with a BamHI digest what would be the Results for X1/X1, X1/X2 and X2/X2 individuals?
If we used probe2 for southern blots with a EcoRI digest what would be the results for X1/X1, X1/X2 and X2/X2 individuals?
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RFLP
RFLP’s are found by trial and error and they require an appropriate probe and enzymeThey are very valuable because they can be used just like any other genetic marker to map genes
They are employed in recombination analysis (mapping) in the same way as conventional Allelic variants are employed
The presence of a specific restriction site at a specific locus on one chromosome and its absence at a specific locus on another chromosome can be viewed as two allelic forms of a gene
The phenotype in this case is a Southern blot rather than white eye/red eye
Lets review standard mapping:To map any two genes with respect to one another, they must be heterozygous at both loci.
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Mapping
Gene W and B are responsible for wing and bristle development
Centromere TelomereW B
To find the map distance between these two genes we need allelic variants at each locus
W=wings B=Bristlesw= No wings b= no bristles
To measure genetic distance between these two genes, the double heterozygote is crossed to the double homozygote
MappingBoth the normal and mutant alleles of gene B (B and b) are sequenced and we find
Centromere TelomereW B
GAATTC
AAATTC
By chance, this mutation disrupts the amino acid sequence and also a EcoRI site!
If DNA is isolated from B/B, B/b and b/b individuals, cut with EcoRI and probed in A Southern blot, the pattern that we will obtain will be
B2 3
E E E
b5E E
B/B Bristle B/b Bristle b/b No bristle
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Mapping
Therefore in the previous cross (WB/wb x wb/wb), the genotype at the B locus can be distinguished either by the presence and absence of bristles or Southern blots
WB/wb x wb/wbFemale Male
Wings No wingsBristles No Bristles
Southern blot: Southern blot:
5 and 2 kb band 5 kb band
There are some phenotypes for specific genes that are very painful to measure
Having a RFLP makes the problem easier
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Mapping
The same southern blot method can be employed for the (W) wing Locus with a different restriction enzyme (BamHI) if an RFLP exists at this locus !!You make the DNA, digest half with EcoRI and probe with bristle probeDigest the other half with BamHI and probe with the wing probe.
GTATCC
GGATCC
W8
B B
wB BB
4 4
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Mapping
To find the map distance between genes, multiple alleles are required.
We can determine the distance between W and B by the classical Method because multiple alleles exist at each locus (W & w, B & b)
You find a new gene C. There are no variants of this gene that alter the phenotype of the fly, that you can observe. Say we don’t even know the function of this gene. You can’t even predict its phenotype.
However the researcher identified an RFLP variant in this gene.
Centromere
TelomereW B C R
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MappingC
c
8E E
26E E E
With this RFLP, the C gene can be mapped with respect to other genes:
Genotype/phenotype relationships for the W and C genes
To determine map distance between R and C, the following cross is performed
W C w c------------ ------------------------ ------------w c w c
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Mapping
Male gamete (wc)
Fema
le gam
ete
W B C RW C(8)
w c(6,2) w c(6,2)
w c(6,2)
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Mapping
Prior to RFLP analysis, only a few classical markers existed in humans
Now over 7000 RFLPs have been mapped in the human genome.
Newly inherited disorders are now mapped by determining whether they are linked to previously identified RFLPs
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Genetic polymorphism
•Genetic Polymorphism: A difference in DNA sequence among individuals, groups, or populations.
•Genetic Mutation: A change in the nucleotide sequence of a DNA molecule.
Genetic mutations are a kind of genetic polymorphism.
Single nucleotidePolymorphism(point mutation)
Repeat heterogeneity
Genetic Variation
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SNP
•A Single Nucleotide Polymorphism is a source variance in a genome. •A SNP ("snip") is a single base mutation in DNA.
•SNPs are the most simple form and most common source of genetic polymorphism in the human genome (90% of all human DNA polymorphisms).
•There are two types of nucleotide base substitutions resulting in SNPs:
–Transition: substitution between purines (A, G) or between pyrimidines (C, T). Constitute two thirds of all SNPs.
–Transversion: substitution between a purine and a pyrimidine.
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SNPs- Single Nucleotide Polymorphisms
Instead of using restriction enzymes, these are found by direct sequencingThey are extremely useful for mapping
Markers
Classical Mendelian 100RFLPs 7000SNPs 1.4x106
-----------------------ACGGCTAA
-----------------------ATGGCTAA
SNPs occur every 300-1000 bp along the 3 billion long human genome
Many SNPs have no effect on cell function
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SNPs
Humans are genetically >99 per cent identical: it is the tiny percentage that is different
Much of our genetic variation is caused by single-nucleotide differences in our DNA : these are called single nucleotide polymorphisms, or SNPs. As a result, each of us has a unique genotype that typically differs in about three million nucleotides from every other person.
SNPs occur about once every 300-1000 base pairs in the genome, and the frequency of a particular polymorphism tends to remain stable in the population.
Because only about 3 to 5 percent of a person's DNA sequence codes for the production of proteins, most SNPs are found outside of "coding sequences".
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SNPs, RFLPs, point mutations
GAATTC GAATTC GAATTC GAATTC GAATTC GAATTC
GAATTC GAATTC GAGTTC GAATTC GAATTCGACTTC
Pt mutSNP
RFLPSNP
RFLPPt mutSNP
SNP
Coding Region SNPs
Occasionally, a SNP may actually cause a disease.SNPs within a coding sequence are of particular interest to researchers because they are more likely to alter the biological function of a protein.
•Types of coding region SNPs–Synonymous: the substitution causes no amino acid change to the protein it produces. This is also called a silent mutation.
–Non-Synonymous: the substitution results in an alteration of the encoded amino acid. A missense mutation changes the protein by causing a change of codon. A nonsense mutation results in a misplaced termination.
–One half of all coding sequence SNPs result in non-synonymous codon changes.
Intergenic SNPs
Researchers have found that most SNPs are not responsible for a disease state because they are intergenic SNPs
Instead, they serve as biological markers for pinpointing a disease on the human genome map, because they are usually located near a gene found to be associated with a certain disease.
Scientists have long known that diseases caused by single genes and inherited according to the laws of Mendel are actually rare.
Most common diseases, like diabetes, are caused by multiple genes. Finding all of these genes is a difficult task.
Recently, there has been focus on the idea that all of the genes involved can be traced by using SNPs.
By comparing the SNP patterns in affected and non-affected individuals—patients with diabetes and healthy controls, for example—scientists can catalog the specific DNA variations that underlie susceptibility for diabetes
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PCR
If a region of DNA has already been cloned and sequenced, the sequence can be used to isolate and amplify that sequencefrom other individuals in a population.
Individuals with mutations in p53 are at risk for colon cancer
To determine if an individual had such a mutation, prior to PCROne would have to clone the gene from the individual of interest(construct a genomic library, screen the library, isolate the Clone and sequence the gene).
With PCR, the gene can be isolated directly from DNA isolated from that individual.
No lengthy cloning procedure
Only small amounts of genomic DNA required
30 rounds of amplification can give you >109 copies of a gene
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PCR
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PCR
Genotype and HaplotypeIn the most basic sense, a haplotype is a “haploid genotype”.
Haplotype: particular pattern of sequential SNPs (or alleles) found on a single chromosome in a single individual
The DNA sequence of any two people is 99 percent identical.
Sets of nearby SNPs on the same chromosome are inherited in blocks. Blocks may contain a large number of SNPs, but a few SNPs are enough to uniquely identify the haplotypes in a block.
The HapMap is a map of these blocks and the specific SNPs that identify the haplotypes are called tag SNPs.
Haplotyping: involves grouping individuals by haplotypes, or particular patterns of sequential SNPs, on a single chromosome.
Microarrays, and sequencing are used to accomplish haplotyping.
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SNP mapping is used to narrow down the known physical location of mutations to a single gene.
The human genome sequence provided us with the list of many of the parts that make a human.
The HapMap provides us with indicators which we can focus on in looking for genes involved in common disease.
Using the HapMap data we compare the SNP patterns of people affected by a disease with those of unaffected people.This allows researchers to survey the whole genome quickly and identify genetic contributions to common diseases--the HapMap Project has simplified the search for gene variants.
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A recessive disease pedigree
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Mapping recessive disease genes with DNA markers
DNA markers are mapped evenly across the genomeThe markers are polymorphic- they look slightly different in Different individuals.We can tell looking at a particular individual which grandparent Contributed a certain part of its DNA. If we knew that grandparent carried the disease, we could sayThat part of the DNA might be responsible for the disease.
A B C D E F G H I
4 different alleles at each locus
A1, A2, A3, A4
B1, B2, B3, B4
C1, C2,………….
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Mapping recessive disease genes with DNA markers
Grandparents 1 and 4 and offspring 1 and 4 have a disease We would look at the markers and see that ONLY at position G do offspring 1 and 4 have the DNA from grandparents 1 and 4.It is therefore likely that the disease gene will be somewhere near marker G.
A B C D E F G H I
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Genetic polymorphism
•Genetic Polymorphism: A difference in DNA sequence among individuals, groups, or populations.
•Genetic Mutation: A change in the nucleotide sequence of a DNA molecule.
Genetic mutations are a kind of genetic polymorphism.
Single nucleotidePolymorphism(point mutation)
Repeat heterogeneity
Genetic Variation
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Repeats
Variation between people- small DNA change – a single nucleotide polymorphism [SNP] – in a target site, RFLPs and SNPs are proof of variation at the DNA level,
Satellite sequences: a short sequence of DNA repeated many times in a row.
Chr1
Chr2
Interspersed
tandem
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RepeatsSatellite sequences: a short sequence of DNA repeated many times in a row.
Chr1
Chr2
Interspersed
tandem
E E E
2 5 6
EE E3 1 4 0.5
1
3
5
Repeat probe
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Repeat expansion
Tandem repeats expand and contract during recombination. Mistakes in pairing leads to changes in tandem repeat numbers
E E4
E1
EE2
EE2
Individual 1
EE3
Individual 2
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Micro-satellite
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DNA finger printing
Variation between people- small DNA change – a single nucleotide polymorphism [SNP] – in a target site, RFLPs and SNPs are proof of variation at the DNA level,
Satellite sequences: a short sequence of DNA repeated many times in a row.
September 1984, Make a probe that hybridizes to these minisatellites at the same time.Hybridize the probe to a blot with DNA from several different people.The X-ray of the blot was developed in Leicester University. 'what a complicated mess’ says Professor Jeffreys, then suddenly he realized -we had patterns”
"There was a level of individual specificity”
Class size No of loci method
SNP 1 bp 100 million PCR/microarray
Micro ~100bp 200,000 PCR
Mini 1-20kb 30,000 southern blot
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DNA fingerprint
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.The use of microsatellite analysis in genetic profiling. In this example, 2 different microsatellites located on the short arm of chromosome 6 have been amplified by the polymerase chain reaction (PCR). The PCR products are labeled with a blue or green fluorescent marker and run in a polyacrylamide gel each lane showing the genetic profile of a different individual. Each individual has a different genetic profile because each person has a different set of microsatellite length variants, the variants giving rise to bands of different sizes after PCR.