dna sequencing 8.2. dna sample in order to sequence or analyze dna, more than just a small sample is...
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DNA Sequencing
8.2
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DNA sample
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• in order to sequence or analyze DNA, more than just a small sample is needed
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Polymerase Chain Reaction (PCR)
• a direct method of making many copies of a DNA sequence
• each cycle doubles the number of DNA molecules (exponential increase)
• useful in forensics, medical diagnosis and genetic research because a small amount of DNA can be amplified
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Taq polymerase
• from Thermus aquaticus (bacterium that lives in hot springs)
• not denatured at the high temperatures needed in PCR
• works optimally at 72ºC
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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” (one of my favourites):
http://learn.genetics.utah.edu/content/labs/pcr/
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Gel Electrophoresis
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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
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Gel Electrophoresis
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Gel Electrophoresis
• Another excellent interactive animation:http://learn.genetics.utah.edu/content/labs/gel/
• Step-by-step slideshow on gel electrophoresis:
http://www.dnalc.org/resources/animations/gelelectrophoresis.html
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DNA Sequencing
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DNA Sequencing
• chain termination method (Sanger dideoxy method) developed in the 1970s by Frederick Sanger
• Human Genome Project used 2 methods of sequencing, both involving the Sanger method
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Deoxy- vs. Dideoxy
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Sanger method
Excellent simple narrated animation (the link doesn’t seem to work, but if you cut & paste the address it does…strange):
http://smcg.ccg.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf
McGraw-Hill animation & quiz:http://highered.mcgraw-hill.com/sites/
0072556781/student_view0/chapter15/animation_quiz_1.html
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Whole-Genome Shotgun
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So you’ve sequenced it…
…what can you do with the data?
• structural genomics
• functional genomics
• bioinformatics
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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)
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$1000 Genome Initiative• currently: about 8 days
and $10 000 to sequence the human genome
• the race is on to be able to sequence the genome faster and for less than $1000
• nanopore sequencing
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DNA Microarray
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DNA Microarray
• allows scientists to pinpoint the functions of specific genes
• the expression of thousands of genes in a particular cell can be identified simultaneously
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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
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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
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Microarray Animation
• Excellent step-by-step animation with simplified explanation of process:
http://www.bio.davidson.edu/courses/genomics/chip/chipQ.html