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 3x109 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

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
2
Mutant GAGTTC
 RFLP

The internal EcoRI site is missing in the second individual

For X1 the sequence at this site is GAATTC

CTTAAG

This is the sequence recognized by EcoRI

The equivalent site in the X2 individual is mutated

GAGTTC
CTCAAG

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 blots

In the region of the human X chromosome, two forms of the
X-chromosome are Segregating in the population.

X1

B R R R B R
4 5 3 6 3.5
2
1

Digest DNA with

EcoRI and probe with
probe1
What do we get?

X2

B R R B R
4 8 6 3.5 4
2
1
 RFLP
Digesting with BamHI and performing Southern blots with the above
probe produces the following results:

X1

B R R R B R
4 5 3 6 3.5

X2

B R R B R
4 8 6 3.5
2
1

There is no variation with respect to the BamHI sites, all

individuals produce the same banding patterns on Southern blots

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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?
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18
18

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&3
8, 5 & 3
8

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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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 Mapping
Lets 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

W B
Centromere Telomere

To find the map distance between these two genes we need allelic
variants at each locus

W=wings B=Bristles
w= 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)

Female gamete

Map distance= # recombinants /Total progeny

7/101= 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

W B
Centromere Telomere

B GAATTC
2 3
E E E

b
AAATTC
E 5 E

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

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/wb
Female Male

Wings No wings
Bristles 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 51
5kb 2kb
Female gamete

wb wb/wb No wings 43
5kb

Recombinant

Wb Wb/wb Wings 3
5kb

wB wB/wb No wings 4
5kb 2kb

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Map distance= # recombinants /Total progeny 7/101= 7 M.U.
 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
probe
Digest the other half with BamHI and probe with the wing probe.

W
8 GTATCC
B B

w
4 4 GGATCC
B B B

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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)

W B C
Centromere R Telomere
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.

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 Mapping
C E 8 E

c 6 2
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 eyes

ww = white eyes

CC = 8kb band
C/c = 8, 6, 2 kb bands
cc = 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

W B C
R
W C(8) w c(6,2)

w c(6,2) w c(6,2)

Male gamete (wc)

Female gamete

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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.

Genetic Variation

Single nucleotide Repeat heterogeneity

Polymorphism
(point mutation)

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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

-----------------------ACGGCTAA

-----------------------ATGGCTAA

Instead of using restriction enzymes, these are found by direct

sequencing
They are extremely useful for mapping

Markers

Classical Mendelian ~200

RFLPs 7000
SNPs 1.4x106

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 progeny

Haplotype1 (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 GACTTC

RFLP RFLP
SNP Pt mut
Pt mut SNP
SNP 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.
 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
 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

WT Mut

500

400

300

200

100
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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.
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.

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 T
Position3 ………………..

Grand
parent 1 2 3 4

A-A-A-A-A-A-A-A-A C-C-C-C-C-C-C-C-C G-G-G-G-G-G-G-G-G T-T-T-T-T-T-T-T-T

Chromosome A-A-A-A-A-A-A-A-A C-C-C-C-C-C-C-C-C G-G-G-G-G-G-G-G-G T-T-T-T-T-T-T-T-T

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 1 2 3 4 5 6 7 8 9

Grand-parent 1 2 3 4
A-A-A-A-A-A-A-A-A C-C-C-C-C-C-C-C-C G-G-G-G-G-G-G-G-G T-T-T-T-T-T-T-T-T
A-A-A-A-A-A-A-A-A C-C-C-C-C-C-C-C-C G-G-G-G-G-G-G-G-G T-T-T-T-T-T-T-T-T

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Haplotyping with microarrays

AlleleA AlleleB

SNP SNP

Design 20mer oligonucleotide probes complementary to the

Polymorphisms

The probes are arrayed on a slide

Each spot corresponds to a polymorphism

Isolate DNA

Label DNA and hybridize to array

Labeled Chromosomal 20mer probe

Hybridization
signal

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.

Genetic Variation

Single nucleotide Repeat heterogeneity

Polymorphism
(point mutation)

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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

Interspersed

Chr2
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)

E E E E
2 5 6
Chr1

Chr2
3 1 4
tandem 0.5

E E E E

1 37
Repeat probe
 Repeat expansion

Tandem repeats expand and contract during recombination.

Mistakes in pairing leads to changes in tandem repeat numbers
These can be detected by Southern blotting

Individual 1

E E
2

Individual 2

E E
3
Ind2
Ind1

5
There are on average
between 2 and 10 alleles
3 (repeats) per mini-sat locus

1
38
Micro-satellite and PCR

39
 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.

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)

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

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