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 genome- the 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’SSHOTGUNNING 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