Download RFLP

RFLP analysis
RFLP= Restriction fragment length polymorphism

Refers to variation in restriction sites between individuals in a population
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Acu pada variasi di (dalam) lokasi pembatasan antar[a] individu di (dalam) suatu populasi

These are extremely useful and valuable for geneticists (and lawyers)

Ini [yang] berharga dan bermanfaat untuk ahli genetika ( dan para pengacara)

On average two individuals (humans) vary at 1 in 1000 bp

Pada [atas] rata-rata dua individu ( manusia) bertukar-tukar pada 1 1000 bp

The human genome is 3x109 bp

Manusia [itu] Genome adalah 3x109 bp
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This means that they will differ in more than 3 million bp.

[Alat/ makna] ini yang mereka akan berbeda lebih dari 3 juta bp
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By chance these changes will create or destroy the recognition sites for Restriction enzymes

Kebetulan perubahan ini akan menciptakan atau menghancurkan lokasi pengenalan [itu] untuk Enzim Pembatasan
1
RFLP
Lets generate a restriction map for a region of human X-chromosome
Biarkan menghasilkan suatu peta pembatasan untuk suatu daerah manusia XChromosome
The restriction map in the same region of the X chromosome of a second individual
may appear as
Pembatasan memetakan di [dalam] daerah sama X chromosome suatu detik/second
individu boleh nampak [ketika;seperti]
Normal
GAATTC
Mutant
GAGTTC
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RFLP
The internal EcoRI site is missing in the second individual
Ecori Yang internal Lokasi adalah hilang individu yang kedua
For X1 the sequence at this site is GAATTC
This is the sequence recognized by EcoRI
CTTAAG
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.
Sekarang jika kita menguji sejumlah besar manusia pada lokasi ini [yang] kita boleh temukan bahwa 25%
menguasai Ecori [itu] Lokasi dan 75% kekurangan lokasi ini
We can say that a restriction fragment length polymorphism exits in this region
Kita dapat kata[kan bahwa suatu panjangnya fragmen pembatasan polymorphism pergi daerah ini
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.
Ini polymorphisms pada umumnya tidak mempunyai manapun konsekwensi phenotypic
Mutasi diam yang tidak mengubah urutan protein [itu] oleh karena pemborosan di (dalam) Codon Pemakaian,
Lokalisasi ke introns atau daerah non-genic atau tidak mempengaruhi 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.
Digest DNA with
EcoRI and probe with
probe1
What do we get?
4
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:
Pencernaan dengan Bamhi dan melakukan/menyelenggarakan Selatan menodai dengan di
atas pemeriksaan menghasilkan hasil yang berikut untuk X1/X1, X1/X2 Dan X2/X2
Individu:
There is no variation with respect to the BamHI sites, all
individuals produce the same banding patterns on Southern blots
Tidak ada variasi berkenaan dengan Bamhi Lokasi, semua
individu menghasilkan yang sama menjilid pola teladan pada [atas] Titik noda
Selatan
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?
Jika kita probe2 digunakan untuk selatan menodai dengan suatu Bamhi mencerna
apa [yang] akan adalah Hasil untuk X1/X1, X1/X2 Dan X2/X2 Individu?
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?
Jika kita probe2 digunakan untuk selatan menodai dengan suatu Ecori mencerna
apa [yang] akan adalah hasil untuk X1/X1, X1/X2 Dan X2/X2 Individu?
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RFLP
RFLP’s are found by trial and error and they require an appropriate probe and enzyme
They are very valuable because they can be used just like any other genetic marker to map genes
RFLP’S ditemukan oleh mencoba-coba dan mereka memerlukan suatu enzim dan pemeriksaan sesuai
Mereka adalah sangat bernilai sebab mereka dapat digunakan seperti halnya penanda [yang] hal azas keturunan lain
untuk memetakan gen
They are employed in recombination analysis (mapping) in the same way as conventional Allelic variants are employed
Mereka dipekerjakan analisa penggabungan-ulang ( pemetaan) dengan cara yang sama seperti Varian [yang] Allelic
konvensional dipekerjakan
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
Kehadiran suatu lokasi pembatasan spesifik pada suatu tempat spesifik pada [atas] satu chromosome dan
ketidakhadiran nya pada suatu tempat spesifik pada [atas] chromosome lain dapat dipandang sebagai dua format
[yang] allelic suatu gen
The phenotype in this case is a Southern blot rather than white eye/red eye
Phenotype dalam hal ini adalah suatu Selatan menodai dibanding/bukannya mata eye/red putih
Lets review standard mapping:
To map any two genes with respect to one another, they must be heterozygous at both loci.
Biarkan standard tinjauan ulang [yang] memetakan:
Untuk memetakan manapun dua gen berkenaan dengan satu sama lain, mereka harus heterozygous pada keduaduanya loci.
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Mapping
Gene W and B are responsible for wing and bristle development
Centromere
W
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
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Mapping
Both the normal and mutant alleles of gene B (B and b) are
sequenced and we find
Centromere
W
B
E
B
2
Telomere
3
E
GAATTC
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
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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
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
9
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
B
w
B
4
B
B
4
B
GTATCC
GGATCC
10
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.
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Mapping
C
E
c
8
6
E
With this RFLP, the C gene can be mapped with respect to
other genes:
E
E
2
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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E
Mapping
W
B
C
W
C(8)
w
w
c(6,2)
w
R
c(6,2)
c(6,2)
Female gamete
Male gamete (wc)
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SNPs, RFLPs, point mutations
GAATTC
GAATTC
GAATTC
GAATTC
SNP
GAATTC
GAATTC
GAATTC
GAGTTC
RFLP
SNP
Pt mut
SNP
GAATTC
GAATTC
GAATTC
GACTTC
RFLP
Pt mut
SNP
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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 sequence from other individuals in a population.
Jika suatu daerah DNA telah sequenced dan cloned, urutan dapat digunakan untuk
mengisolasikan dan memperkuat suara urutan itu dari individu lainnya di (dalam) suatu populasi.
Individuals with mutations in p53 are at risk for colon cancer
Individu dengan mutasi di (dalam) p53 berhadapan dengan resiko untuk kanker tanda titik dua
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).
Untuk menentukan jika perorangan telah mutasi seperti itu, sebelum PCR Orang akan harus clone gen dari
individu [bunga/minat] ( membangun suatu perpustakaan genomic, menyaring perpustakaan [itu], mengisolasikan
Clone [itu] Dan Urutan gen).
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
15
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 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.
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
18
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.
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
A
B
C
D
E
F
G
H
I
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.
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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.
Genetic Variation
Single nucleotide
Polymorphism
(point mutation)
Repeat heterogeneity
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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
Interspersed
Chr2
tandem
22
Repeats
Satellite sequences: a short sequence of DNA repeated many times in a row.
2
5
E
6
E
E
Chr1
Interspersed
Chr2
3
E
1
E
tandem
4
E
0.5
5
3
1
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Repeat probe
Repeat expansion
Tandem repeats expand and contract during recombination.
Mistakes in pairing leads to changes in tandem repeat numbers
E
1
E
E
E
2
4
E
Individual 1
E
E
2
Individual 2
E
3
E
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