rflp analysis

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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

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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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Page 1: RFLP analysis

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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

5kb 3kb

The restriction map in the same region of the X chromosome of a second individual may appear as

8kb

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.

B R R R B R

4 5 3 6 3.5

B R R B R

4 8 6 3.5

X1

X2

Digest DNA with EcoRI and probe with probe1What do we get?

21

21

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RFLP

Digesting with BamHI and performing Southern blots with the above probe produces the following results:

There is no variation with respect to the BamHI sites, all individuals produce the same banding patterns on Southern blots

B R R R B R

4 5 3 6 3.5

B R R B R

4 8 6 3.5

X1

X2

21

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RFLP in individuals

If we used probe1 for southern blots with a BamHI digest what would be the Results for X1/X1, X1/X2 and X2/X2 individuals?

181818

If we used probe1 for southern blots with a EcoRI digest what would be the results for X1/X1, X1/X2 and X2/X2 individuals?

5 & 38, 5 & 38

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RFLP

RFLP’s are found by trial and error and they require an

Appropriate probe AND appropriate enzyme

They 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 morphological allele markers 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

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Using RFLPs to map human disease genes

Which RFLP pattern segregates with the diseased individuals

Top or bottom

Using DNA probes for different RFLPs you screen individuals for a RFLP pattern that shows co-inheritance with the disease

Conclusion: the actual mutation resides at or near the RFLP

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MappingLets review standard mapping:To map any two genes with respect to one another, they must be heterozygous at both loci.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

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Mapping

Male gamete (wb)

Fem

ale

gam

ete

Map distance= # recombinants /Total progeny7/101= 7 M.U.

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Mapping

Both 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

Just like Genes, RFLPs mark specific positions on chromosomes and can be for mapping.

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Mapping

Male gamete (wb)

Genotype phenotype

Parental

WB WB/wb Wings 515kb 2kb

wb wb/wb No wings 435kb

Recombinant

Wb Wb/wb Wings 35kb

wB wB/wb No wings 45kb 2kb

Fem

ale

gam

ete

Map distance= # recombinants /Total progeny 7/101= 7 M.U.

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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

Telomere

W B C R

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MappingC

c

8E E

26

E 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

WW and Ww = Red eyesww = white eyes

CC = 8kb bandC/c = 8, 6, 2 kb bandscc = 6, 2 kb bands

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)

Fem

ale

gam

ete

W B C R

W 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 subset 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 change 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.

While a single base can change to all of the other three bases, most SNPs have only one allele.

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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 ~200RFLPs 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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How did SNPs arise?

F2a----ACGGACTGAC----CCTTACGTTG----TACTACGCAT---- |

F1 ----ACTGACTGAC----CCTTACGTTG----TACTACGCAT----

P ----ACTGACTGAC----CCTTACGTTG----TACTACGCAT---- |

F1 ----ACTGACTGAC----CCTTACGTTG----TACTAGGCAT----

| |F2b----ACTGACTGAC----CCATACGTTG----TACTAGGCAT----

Compare the two F2 progenyHaplotype1 (F2a) = SNP allele1

----ACGGACTGAC----CCTTACGTTG----TACTACGCAT----Haplotype2 (F2b) = SNP allele2----ACTGACTGAC----CCATACGTTG----TACTAGGCAT----

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SNPs, RFLPs, point mutations

GAATTC GAATTC GAATTC GAATTC GAATTC

GAATTC GAGTTC GAATTC GAATTC

Pt mutSNP

RFLPSNP

RFLPPt mutSNP

GACTTC

SNP

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Coding Region SNPs

•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.

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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 information can be used to isolate and amplify that sequence from 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 PCR one 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 and RFLP

WT ----------CCTGAGGAG--------------------------GGACTCCTC----------------

MSTII

Mut ----------CCTGTGGAG--------------------------GGACACCTC----------------

PCR amplify DNA from normal and sickle cell patient

Digest with MstII

100

200

300

400

500

WT Mut

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Genotype and Haplotype

In 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 specific SNPs that identify the haplotypes are called tag SNPs. This will make genome scan approaches to finding regions with genes that affect diseases much more efficient and comprehensive.

Haplotyping: involves grouping individuals by haplotypes, or particular patterns of sequential SNPs, on a single chromosome.

There are thought to be a small number of haplotype patterns for each chromosome.

Microarrays, PCR 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 to make a human.

The HapMap provides us with indicators which we can focus on in looking for genes involved in common disease.

By using HapMap data to compare the SNP patterns of people affected by a disease with those of unaffected people, researchers can survey the whole genome and identify genetic contributions to common diseases more efficiently than has been possible without this genome-wide map of variation: the HapMap Project has simplified the search for gene variants.

Oligonucleotide chips contain thousands of short DNA sequences immobilised at different positions. Such chips can be used to discriminate between alternative bases at the site of a SNP.

Chips allow many SNPs to be analyzed in parallel.

Short DNA sequences on the chip represent all possible variations at a polymorphic site;

A labeled DNA will only stick if there is an exact match. The base is identified by the location of the fluorescent signal.

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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 genome. The 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 say that part of the DNA might be responsible for the disease.

Grandparent 1 2 3 4

ChromosomeA-A-A-A-A-A-A-A-AA-A-A-A-A-A-A-A-A

C-C-C-C-C-C-C-C-CC-C-C-C-C-C-C-C-C

G-G-G-G-G-G-G-G-GG-G-G-G-G-G-G-G-G

T-T-T-T-T-T-T-T-TT-T-T-T-T-T-T-T-T

1 2 3 4 5 6 7 8 9

4 different alleles at each locus

Position1 can be A or C or G or T

Position2 can be A or C or G or TPosition3 ………………..

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Grand-parent

1 2 3 4

1 2 3 4 5 6 7 8 9

A-A-A-A-A-A-A-A-AA-A-A-A-A-A-A-A-A

C-C-C-C-C-C-C-C-CC-C-C-C-C-C-C-C-C

G-G-G-G-G-G-G-G-GG-G-G-G-G-G-G-G-G

T-T-T-T-T-T-T-T-TT-T-T-T-T-T-T-T-T

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Haplotyping with microarrays

SNP

Design 20mer oligonucleotide probes complementary to the Polymorphisms

The probes are arrayed on a slideEach spot corresponds to a polymorphism

SNP

Isolate DNA

AlleleA AlleleB

Label DNA and hybridize to array

Labeled Chromosomal 20mer probe

Hybridizationsignal

No signal

There are ~3 thousand different probes per microarray

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Genetic polymorphism

•Genetic Polymorphism: A difference in DNA sequence among individuals, groups, or populations.

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 point mutations are proof of variation at the DNA level.

Satellite sequences: a short sequence of DNA repeated many times.

Chr1

Chr2

Interspersed

tandem

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Mini Satellite Repeats and Blots

Mini Satellite sequences: a short sequence (20-100bp long) of DNA repeated many times (alleles vary in length from 0.5 to 20 kb)

Chr1

Chr2

tandem

E E E2 5 6

EE E

3 1 4 0.5

1

3

5

Repeat probe

E

E

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Repeat expansion

Tandem repeats expand and contract during recombination. Mistakes in pairing leads to changes in tandem repeat numbersThese can be detected by Southern blotting

EE2

Individual 1

EE3

Individual 2

1

3

5

Ind

1

Ind

2

There are on average between 2 and 10 alleles (repeats) per mini-sat locus

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Micro-satellite and PCR

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DNA finger printingVariation 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.

Class size No of loci method

SNP 1 bp 100 million PCR/microarray

Micro ~200bp 200,000 PCR

Mini 0.2-20kb 30,000 southern blot

Micro satellite are 2-4 bp repeats in tandem repeats 15-100 times in a row

Mini satellite are 20-100 bp repeats in tandem (0.5 to 20kb long)