JOS FERNNDEZ PIQUERAS

Catedrtico de Gentica
Universidad Autnoma de Madrid

Leccin 8c
Mapas fsicos
How to identify a gene if we were not aware about its function
The problem:
-You have to localize a friend (gene) living at C/Angel Ganivet (Granada)
-You have to localize a gene in an specific site on the human genome

There are available two categories of maps:

1.- Genetics maps (linkage):

-Interstate highway maps that guide to a city
-Provide and indirect estimate (cM) of the distance between
items (genes or molecular markers)

2.- Physical maps:

-Street maps
-Provide an estimate of the true distance (base-pairs)
2.- Physical maps:
-Street maps
-Provide an estimate
of the true distance
(base-pairs)
Types of physical maps (from low to high resolution):

1.- Chromosomal or cytogenetic maps:

-based on distinctive banding patterns
-FIS (clasical, painting, spectral karyotypes etc.)
-CGH (CGH-arrays)
2.-Somatic cell hybrids/Radiation Hybrid maps)
3.-Collection of overlapping DNA fragments
-Molecular cloning to generate libraries
-Assembly of overlapping clones: clone contigs.
-Sequence tagged sites (STSs) (short unique DNA sequences
from different sources: expressed sequence tags (ESTs), Simple
sequence length polymorphisms (SSLPs), random genomic
sequences etc.
-Restriction fragments
4.- Sequentiacin
1.- Chromosomal
or cytogenetic
maps
14 Banding pattern of human chromosomes
Until 1970s chromsomes were identified on the basis of

Afterwards, the introduction of

BANDEO DE ALTA RESOLUCIN
 G-banding (trypsine + giemsa)
15
16

Craig Venter karyotype (G-banding)

17

FISH
18 Chromosome painting

Chromosome 3: biotine and Cy3; chromosome 9: FITC; chromosome 17:

biotine and FICT.
20
2.-Somatic cell
hybrids/Radiation
Hybrid maps)
 Fibroblastos humanos Fibroblastos de ratn
HPRT-/TK+ HPRT+/TK-

virus Sendai o polietilenglicol

Medio HAT
Lneas celulares hbridas
Colonia hbrida A
A B
Cromosomas humanos 1 + +
2 + -
3 - -
Colonia hbrida B
Genes humanos a + -
b + +

Cartografiado mediante hibridacin celular somtica

En los hbridos celulares se pueden distinguir los cromosomas humanos y los
del roedor

Humanos Roedor

La asignacin de genes en cromosomas humanos se resuelve como un puzzle

Separacin de cromosomas mediante citometra de flujo
Hbridos de
radiacin
3.-Collection of overlapping DNA fragments
-Sequence tagged sites (STSs)
-Restriction fragments
-Molecular cloning to generate libraries
-Assembly of overlapping clones: clone
contigs.
 4.- Secuenciacin: el mapa fsico ms detallado posible
P. Berg, W. Gilbert & F. Sanger, premios Nobel de 1980 por sus estudios sobre el DNA recombinante y el
desarrollo de una metodologa eficaz para la secuenciacin de los cidos nucleicos

P.Berg (1928-)

K.B. Mullis: premio

Nobel de 1993 por el
desarrollo del mtodo
de amplificacin en
cadena de la
polimerasa (PCR)

clonacin de un fragmento de DNA en

una bacteria
25

Methods to Sequencing DNA

1.- Chemical method: Maxan and Gilbert

(1980)

-2.- Enzymatic synthesis and dideoxy

sequencing (the vast majority of current
DNA sequencing): Sanger (1980).

-3.- The pyrosequencing method

26
1.- Chemical method: Maxan and Gilbert
(1977-80):based on chemical modification of DNA
and subsequent cleavage at specific bases
27
2.- The enzymatic method: Sanger
et al.

This page was written by Elizabeth Canfield '99 This figure

was taken with permission from <
http://www.plattsburgh.edu/acadvp/artsci/biology/bio401/
DNASeq.html
28 Automated DNA sequencing
29 3.- Pyrosequencing Technology

From Quiagen
The first generation of DNA sequencing
methodologies

-Started in 1975 with the plus and minus method of

Sanger & Coulson, and in 1977 with the Maxam and
Gilbert chemical method

-the Sanger method allowed reading 80 bases

(enzymatic synthesis using dideoxy-terminators ).

-optimization using fluorescent dideoxynucleotide

dyes and automated detection result in increased
throughput and accuracy, allowing reads of 1000
bases
31 The first draft
Science Feb. 16, 2001 Nature Feb. 15, 2001
(Celera Genomics) (Human Genome Project)
(Craig Venter) (Francis Collins)

Human Genome Sequence from 5 Humans (3 females-2

males) completed
33
Hierarchical shotgun sequencing Whole-genome shotgun sequencing

Cloned to generate a library

In hierarchical shotgun: A library is constructed by fragmenting the target genome and cloning it into a large-fragment cloning
vector; here, BAC vectors are shown. The genomic DNA fragments represented in the library are then organized into a physical
map and individual BAC clones are selected and sequenced by the random shotgun strategy. Finally, the clone sequences are
assembled to reconstruct the sequence of the genome (Nature 2001, 409: 860-925)
Sistemas de secuenciacin de ltima generacin

Strachan & Read (2010) Human Molecular Genetics. Garland. 4th ed.
 Pero:
-La mayora de los genes codificantes humanos pueden producir varios tipos de RNAm y cada
uno de estos varias protenas.
-De la Genmica (21.000 genes) a la Protemica (ms de 200.000 protenas).
-Adems: se siguen descubriendo nuevos genes no-codificantes.
Strachan & Read (2010) Human Molecular Genetics. Garland. 4th ed.
Finis, -is
Fin
Fi
Akabu
Fim

La Fin
Fine
Ende
The End