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
1
RFLP
Lets generate a restriction map for a region of human Xchromosome
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
2
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.
3
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
4
R
R
5
3
B
6
R
3.5
2
1
Digest DNA with
EcoRI and probe with
probe1
What do we get?
X2
B
R
4
R
8
1
B
6
R
3.5
2
4
RFLP
Digesting with BamHI and performing Southern blots with the
above probe produces the following results:
X1
B
R
4
R
5
R
3
B
6
R
3.5
X2
B
R
4
R
8
1
B
6
R
3.5
2
There is no variation with respect to the BamHI sites, all
individuals produce the same banding patterns on Southern
blots
5
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?
18
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
6
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
7
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
8
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
Centromere
B
Telomere
To find the map distance between these two genes we need allelic
variants at each locus
W=wings
w= No wings
B=Bristles
b= no bristles
To measure genetic distance between these two genes, the
double heterozygote is crossed to the double homozygote
9
Mapping
Female gamete
Male gamete (wb)
Map distance= # recombinants /Total progeny
7/101= 7 M.U.
10
Mapping
Both the normal and mutant alleles of gene B (B and b) are
sequenced and we find
W
Centromere
B
B
GAATTC
3
2
E
Telomere
E
E
b
E
5
E
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
B/B Bristle
B/b Bristle
b/b No bristle
11
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
Female
x
wb/wb
Male
Wings
Bristles
No wings
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.
12
Mapping
Male gamete (wb)
Genotype
phenotype
WB
WB/wb
Wings
5kb 2kb
51
wb
wb/wb
No wings
5kb
43
Wb
Wb/wb
Wings
5kb
3
wB
wB/wb
No wings
5kb 2kb
4
Female gamete
Parental
Recombinant
13
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
GTATCC
8
B
w
B
B
4
B
4
B
GGATCC
14
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)
Centromere
W
B
C
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.
15
Mapping
C
c
E
8
E
6
E
2
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
16
Mapping
W
B
C
W
C(8)
w
R
c(6,2)
w
c(6,2)
w
c(6,2)
Female gamete
Male gamete (wc)
17
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
18
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
Polymorphism
(point mutation)
Repeat heterogeneity
19
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.
20
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
RFLPs
SNPs
~200
7000
1.4x106
SNPs occur every 300-1000 bp along the 3 billion long human
genome
Many SNPs have no effect on cell function
21
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".
22
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----
23
SNPs, RFLPs, point mutations
GAATTC
GAATTC
GAATTC
GAATTC
GAGTTC
GAATTC
RFLP
SNP
SNP
Pt mut
SNP
GAATTC
GAATTC
GAATTC
GACTTC
RFLP
Pt mut
SNP
24
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
27
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
28
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.
30
A recessive disease pedigree
31
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
A-A-A-A-A-A-A-A-A
Chromosome A-A-A-A-A-A-A-A-A
2
C-C-C-C-C-C-C-C-C
C-C-C-C-C-C-C-C-C
3
G-G-G-G-G-G-G-G-G
G-G-G-G-G-G-G-G-G
4
T-T-T-T-T-T-T-T-T
T-T-T-T-T-T-T-T-T
32
1
Grand-parent
1
A-A-A-A-A-A-A-A-A
A-A-A-A-A-A-A-A-A
2
2
C-C-C-C-C-C-C-C-C
C-C-C-C-C-C-C-C-C
3 4
5 6
7
3
G-G-G-G-G-G-G-G-G
G-G-G-G-G-G-G-G-G
8 9
4
T-T-T-T-T-T-T-T-T
T-T-T-T-T-T-T-T-T
33
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
34
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
Polymorphism
(point mutation)
Repeat heterogeneity
35
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
36
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
2
E
5
E
6
Chr1
Chr2
3
1
E
E
4
tandem
E
0.5
E
5
3
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
2
E
Individual 2
E
E
Ind2
Ind1
3
5
3
There are on average
between 2 and 10 alleles
(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
40