Human Genome Project Flashcards
In 1970 one DNA fragment of only 30 nucleotides would have taken how long would it take to sequence?
1 year
What was the aim of the Human Genome Project?
Map the entire human genome - determine the complete nucleotide sequence of the DNA of each human chromosome
When was the human genome project proposed?
1985
How much was set aside by NIH and US Dept. of Energy?
$3 billion
What were the two groups called that took part in the human genome project?
USA grp Celera
Sanger Centre UK
What were the 3 stages of the Human Genome project?
Genetic mapping (Linkage mapping) Physical mapping DNA sequencing
What techniques were used in the Human Genome Project?
Use of restriction enzymes and RFLPs
YACs and BACs
PCR and gel electrophoresis
DNA sequencing
Apart for the human genome, which other organisms were also mapped?
Bacteria (E. coli, influenza, several others) Yeast (Saccharomyces cerevisiae) Plant (Arabidopsis thaliana) Roundworm (Caenorhabditis elegans) Fruit fly (Drosophila melanogaster) Mouse (Mus musculus)
What was good to use as a marker during the human genome project?
The markers can be genes or other identifiable DNA sequences such as a RFLPs or short repetitive sequences - microsatellites
How did scientists make sure the DNA fragments were in order during physical mapping?
The trick is to produce restriction fragments that overlap with each other (in other words, 2 fragments will share part of a sequence).
How did scientists get the fragments used for DNA mapping?
DNA fragments for physical mapping are prepared by DNA cloning
With large genomes like the human genome, the researchers carried out several rounds of DNA cutting, cloning and physical mapping.
Smaller and smaller size fragments are used with each round
Why were YAC and BAC used in the cloning of genes for the human genome project?
YAC was used first due to its large plasmid to hold a large number bp
BAC was then used to identify smaller sections of inserted DNA 100k-500K bp.
Explain the Sanger method.
Molecules that are sequenced are cloned restriction fragments (up to 1000bp)
DNA strand to be sequenced is used as a template for new strands (amplified)
Method based on random incorporation of a modified nucleotide
These dideoxynucleotides can terminate a growing DNA chain because they lack a 3’-OH for attachment of the next nucleotide
A series of 4 reaction mixtures for the DNA fragment is prepared
ssDNA is used
Each reaction mixture contains everything needed for synthesis of complementary strand:
Labelled primer known to base-pair with the 3’ end of the strand
DNA polymerase
All four dNTPs present in excess
In addition, each of the 4 reaction mixtures contains a different modified dNTP
This is the basis of the technique - dideoxy-nucleotides ( ddNTPs) are used.
These are similar to dNTPs but terminate a growing DNA chain
They are tagged with fluorescent dyes Each of the 4 ddNTPs is tagged with a different dye.
Once the reaction starts in each mixture, synthesis of a new strand begins.
Recall what happens in a PCR. Under the influence of DNA polymerase, dNTP bases are added to the complimentary strand.
However, once a ddNTP is inserted into a growing DNA strand, synthesis of that strand is stopped immediately
A dideoxynucleotide is inserted at random every so often, instead of its normal dNTP
DNA chain terminated
In this way, a set of strands of various lengths is obtained
All the possible lengths of DNA are represented and every piece of synthesised DNA contains a fluorescent label at its terminus
Amplified DNA can then be separated according to size via gel electrophoresis
As the fluorescent DNA reaches the bottom of the gel (now separated from smallest to largest), a laser can pick up the fluorescence of each piece of DNA.
Sanger method relies on the fact that each ddNTP emits a different fluorescent signal, so that the presence of a ddNTP at the terminus can be recorded on a computer
The reaction is set up so that a fluorescent ddNTP is present at every position in the DNA strand (i.e. every possible size of DNA strand is present)
Every nucleotide in the strand can be determined
The gene has how many bases?
3000
What is the biggest gene and how many bases does it have?
Dystrophin
2.4 million bases
