DNA Sequencing
8.2
Polymerase Chain Reaction (PCR)
• a direct method of making many copies of a DNA sequence
• exponential increase because each cycle doubles the number of DNA molecules
• useful in forensics, medical diagnosis and genetic research because a small amount of DNA can be amplified
Taq polymerase
• from Thermus aquaticus (type of bacteria that live in hot springs)
• is not denatured at the high temperatures needed in PCR
• works optimally at 72ºC
PCR Animations
Look at step-by-step animation of PCR process first:
http://www.dnalc.org/resources/animations/pcr.html
Then this excellent interactive “virtual lab”:http://learn.genetics.utah.edu/content/labs/pcr/
Gel Electrophoresis
Gel Electrophoresis
• a method of separating molecules based on size
• DNA migrates through the gel towards a positive electrode
• smaller fragments move faster through the gel, causing separation by size
• DNA fragments can be seen by using a stain such as ethidium bromide
Gel Electrophoresis
Gel Electrophoresis
• Excellent interactive animation of gel electrophoresis:
http://learn.genetics.utah.edu/content/labs/gel/
• Slideshow with focus on scientists and history of gel electrophoresis:
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
DNA Sequencing
• chain termination method (Sanger dideoxy method) was developed in the 1970s by Frederick Sanger
• Human Genome Project used 2 methods of sequencing, both involving the Sanger method
Deoxy- vs. Dideoxy
Sanger method
Excellent simple narrated animation:http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf
So you’ve sequenced it…
…what can you do with the data?
• structural genomics• functional genomics• bioinformatics
Interesting findings…
• can sequence a gene and then use computer to find similar sequences in the same genome or in other genomes
• have discovered many genes of unknown function
• unexpected degree of similarity between different organisms (e.g., yeast & humans)
$1000 Genome Initiative• it currently takes about 8
days and $10 000 to sequence the human genome
• the race is on to be able to sequence the genome for less than $1000
• nanopore sequencing
DNA Microarray
DNA Microarray
• allows scientists to pinpoint the functions of specific genes
• the expression of thousands of genes in a particular cell can be identified simulaneously
Microarray
• microarray or gene chip can hold from 10 to 2.1 million DNA samples (probes)
• could be used to compare a “normal” cell with a cancerous cell
• isolate mRNA from both cells; synthesize labelled cDNA; denature and place on microarray
DNA Microarray
To read the results:• RED shows gene is expressed in
experimental (cancerous) cell• GREEN show gene is expressed in
normal cell• YELLOW shows gene is expressed in
both cells
Microarray Animation
• Excellent interactive animation with simplified explanation of process:
http://www.bio.davidson.edu/courses/genomics/chip/chipQ.html
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