MOLECULAR DIAGNOSTICS; EXAM 2
Q&A
How do the following more recent projects build on the Human Genome Project:
Hapmap - ANSWER-A public database cataloging SNPs in the human genome, with
the purpose of discovering which are related to specific disease states
How do the following more recent projects build on the Human Genome Project:
1000 genome project - ANSWER-The aligning of 1000+ complete human genome
sequences to gain a more complete understanding of the genetic variability (SNPs)
between individuals.
List some of the major findings of the human genome project. - ANSWER-Size: 2.91
billion base pairs
Number of genes: 30,000 - much fewer than expected
Only 2% sequences codes for genes
Genes are clustered in regions
30-40% of genome has repeat sequences
To improve the Sanger Method, we - ANSWER-1. threw out the polyacrylamide gel,
and use a column instead
2. added fluorescent dyes
3. software to analyze it
Sanger Sequence Machine Steps - ANSWER-1) extract DNA
2) PCR run
3) take product and put on sequencing machine
4) computer generates the data
Why are longer bp sequences better than short ones? - ANSWER-The longer they
are the easier it is to piece them back together
To analyze the data the computer gives you: - ANSWER-copy/paste in database
(BLAST)
- it tells you what it matches
- aligns your sequence with possible matches
Southern hybrization process - ANSWER-1. DNA extraction
2. cut with restriction enzyme
3. Run agarose gel
(depurinate/denature)
4. Transfer DNA from gel to solid support (nitrocellulose membrane)
5. Hybridize probe
6. see what band lit up (detect probe)
How long does a southern take ? - ANSWER-68 degrees C for 5-16 for hybridization
VERY LONG PROCESS so it is not performed in the clinical lab (2.5 days)
, What must you do before putting the sample on the membrane in southern? -
ANSWER-1. depurinate in HCl
2. Denature
How is Northern Hybridization different than Southern? - ANSWER-same
mechanism except the first step..
RNA is extracted instead
the band will be much smaller on the gel
Dot/Slot Blot - ANSWER-a variation of southern hybridization because you dont
necessarily care where you bands sits, just if it is exists
Dot/Slot Blot advantages - ANSWER-cuts off a day and half of work
much faster but gives you less info
** Ideal for multiple samples
Dot/Slot disadvantages (data results) - ANSWER-there are a lot of gray areas
because of the lightness of some bands
Probes - ANSWER-can be DNA or RNA
long pobes of 1000 bp
heat to 95 degree C 10-15 mins before
What are the standard PCR contents? - ANSWER-1) template DNA
2) buffer
3) 2 primers
4) dNTP
5) Taq Polymerase
What are the cycle Sequencing PCR contents - ANSWER-1) template DNA
2) buffer
3) 1 primer (drives the region were sequencing)
4) dNTP and ddNTP
5) Taq DNA Polymerase
What is the purpose of hybridization? - ANSWER-finding DNA sequences
What are the steps of southern hydribization? - ANSWER-1. extraction
2. restriction digestion
3. run on agrose
4. depurinate/denature
5. transfer to nitrocellulose
6. hybridize probe
7. visualize
Why are DNA/RNA immobilized on an inert support (membrane) ? - ANSWER-so
that self-annealing
What does "in-silica hydribizations" refer too? - ANSWER-done on glass slides
Q&A
How do the following more recent projects build on the Human Genome Project:
Hapmap - ANSWER-A public database cataloging SNPs in the human genome, with
the purpose of discovering which are related to specific disease states
How do the following more recent projects build on the Human Genome Project:
1000 genome project - ANSWER-The aligning of 1000+ complete human genome
sequences to gain a more complete understanding of the genetic variability (SNPs)
between individuals.
List some of the major findings of the human genome project. - ANSWER-Size: 2.91
billion base pairs
Number of genes: 30,000 - much fewer than expected
Only 2% sequences codes for genes
Genes are clustered in regions
30-40% of genome has repeat sequences
To improve the Sanger Method, we - ANSWER-1. threw out the polyacrylamide gel,
and use a column instead
2. added fluorescent dyes
3. software to analyze it
Sanger Sequence Machine Steps - ANSWER-1) extract DNA
2) PCR run
3) take product and put on sequencing machine
4) computer generates the data
Why are longer bp sequences better than short ones? - ANSWER-The longer they
are the easier it is to piece them back together
To analyze the data the computer gives you: - ANSWER-copy/paste in database
(BLAST)
- it tells you what it matches
- aligns your sequence with possible matches
Southern hybrization process - ANSWER-1. DNA extraction
2. cut with restriction enzyme
3. Run agarose gel
(depurinate/denature)
4. Transfer DNA from gel to solid support (nitrocellulose membrane)
5. Hybridize probe
6. see what band lit up (detect probe)
How long does a southern take ? - ANSWER-68 degrees C for 5-16 for hybridization
VERY LONG PROCESS so it is not performed in the clinical lab (2.5 days)
, What must you do before putting the sample on the membrane in southern? -
ANSWER-1. depurinate in HCl
2. Denature
How is Northern Hybridization different than Southern? - ANSWER-same
mechanism except the first step..
RNA is extracted instead
the band will be much smaller on the gel
Dot/Slot Blot - ANSWER-a variation of southern hybridization because you dont
necessarily care where you bands sits, just if it is exists
Dot/Slot Blot advantages - ANSWER-cuts off a day and half of work
much faster but gives you less info
** Ideal for multiple samples
Dot/Slot disadvantages (data results) - ANSWER-there are a lot of gray areas
because of the lightness of some bands
Probes - ANSWER-can be DNA or RNA
long pobes of 1000 bp
heat to 95 degree C 10-15 mins before
What are the standard PCR contents? - ANSWER-1) template DNA
2) buffer
3) 2 primers
4) dNTP
5) Taq Polymerase
What are the cycle Sequencing PCR contents - ANSWER-1) template DNA
2) buffer
3) 1 primer (drives the region were sequencing)
4) dNTP and ddNTP
5) Taq DNA Polymerase
What is the purpose of hybridization? - ANSWER-finding DNA sequences
What are the steps of southern hydribization? - ANSWER-1. extraction
2. restriction digestion
3. run on agrose
4. depurinate/denature
5. transfer to nitrocellulose
6. hybridize probe
7. visualize
Why are DNA/RNA immobilized on an inert support (membrane) ? - ANSWER-so
that self-annealing
What does "in-silica hydribizations" refer too? - ANSWER-done on glass slides
