Transcript GENE SEQUENCING - Amirkabir University of Technology

GENE SEQUENCING
INTRODUCTION
CELL
• The cells contain the
nucleus.
• The chromosomes are
present within the
nucleus.
CHROMOSOMES
• Chromosomes are made
up of DNA and Protein.
• The number of
chromosomes varies
among different
organisms.
• Closely related species
have a similar number of
chromosomes- Eg.
Chimpanzees – 48
chromosomes
DNA
• Hereditary material in
all living cells.
• DNA molecule is a
double helix, a
structure that looks
like a ladder twisted
into a spiral.
GENE
• Unit of hereditary
information.
• A section of a DNA
molecule that specifies the
production of a particular
protein
• Different genes determine
the different
characteristics , or traits of
an organism.
PARTS OF A GENE
GENE SEQUENCING
• Gene sequencing is figuring out the order of
DNA nucleotides, or bases, in a genomethe order of As, Cs, Gs, and Ts that make up
an organism’s DNA.
• The human genome is made up of over 3
billion of these genetic letters.
DNA SEQUENCE
• In this sequence an
adenine (A) is
followed by a guanine
(G), which is followed
by a thymine (T),
which in turn is
followed by a cytosine
(C) , another cytosine
and so on.
SEQUENCE
• Buzz word for structure of a DNA molecule
• “ to sequence” – To determine the sequence
of nucleotides it contains
CLONING
• “Clone” – produce copies of it
• Collection of cells containing identical
genetic material
• Clones are produced from a single parent
cell
CLONING
PCR
(Polymerase chain reaction)
• Technique for quickly making many copies of a
specific segment of DNA
• Primers are added (initiate copying of strands)
along with nucleotides and Taq polymerase
• By cycling temperature target DNA is repetitively
denatured and copied
• A single copy can be amplified to obtain billions
of replicates
PCR
PCR
PCR
GEL ELECTROPHORESIS
• Method to analyze size of DNA fragments
• In an electric field larger fragments of DNA
move through a gel slower than smaller
ones.
• Produce migrating bands
• Visualized by soaking gel in a dye
(ethidium bromide)
• DNA flouresce under UV light
SANGER’S TECHNIQUE
• COPYING STRANDS OF DNA WITH
dNTP AND TERMINATING WITH
TAGGED ddNTPs
• SHOWS LOCATION OF NUCLEOTIDES
BY USE OF X-RAY MACHINES
• SLOW AND TEDIOUS
CRAIG VENTER’S
SHOTGUNNING METHOD
• Practice of randomly clipping a larger DNA
fragment into various smaller pieces,
cloning everything and studying resulting
individual clones
• By finding out how regions of subclones
overlap, the sequence of the larger fragment
becomes apparent
DNA SEQUENCING
• Chromosomes, which range in size from 50
million to 250 million bases, must first be broken
into much shorter pieces (subcloning step)
• Each short piece is used as a template to generate
a set of fragments that differ in length from each
other by a single base that will be identified in a
later step (template preparation and sequencing
reaction steps).
DNA SEQUENCING
• The fragments in a set are separated by gel
electrophoresis (separation step).
• New fluorescent dyes allow separation of all four
fragments in a single lane on the gel.
• The final base at the end of each fragment is
identified (base-calling step). This process
recreates the original sequence of As, Ts, Cs, and
Gs for each short piece generated in the first step.
DNA SEQUENCING
• After the bases are "read," computers are used to
assemble the short sequences (in blocks of about
500 bases each, called the read length) into long
continuous stretches that are analyzed for errors,
gene-coding regions, and other characteristics.
• Finished sequence is submitted to major public
sequence databases, such as GenBank.
SEQUENCING
CAPILLARY SEQUENCERS
• The newer machines in
which the DNA is run
through an array of 96 gel
filled capillaries – rather
than a slab of gel.
• Capillary machines read
the base sequence as DNA
moves through the gel.
Lab technician working with
sequencing machines