Molecular Diagnostics; Exam 2
Complete Answers
When is Sanger Sequencing typically used? - ANSWER-It is not used for routine
bacterial diagnosis. It is only used for new/emerging diseases.
What is the typical run length? - ANSWER-500-700 bp
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
A micoarray is testing for: - ANSWER-a lot of genetic characteristics from one
sample
Southern Hybridization - ANSWER-extract DNA
probe w/ DNA
Northern Hybridization - ANSWER-extract RNA
probe w/ DNA
Western Hybridization - ANSWER-extract proteins
probe with antibodies
What is the first method that became standard in DNA sequencing? - ANSWER-
Sanger method
What type of PCR does Sanger Method use? - ANSWER-cycle sequencing PCR
Cycle Sequencing PCR requirements - ANSWER-1. template DNA
2. buffer
3. 1 primer (drives the region we sequence)
4. dNTPs and ddNTPs (the label)
5. Taq DNA Polymerase
Sanger procedure; current method - ANSWER-1. DNA extraction
2. PCR run
3. Take product and and put on sequencing machine
4. computer generates data
What is another name for the Sanger method? - ANSWER-Dideoxy- chain
termination
In what two ways is this "cycle sequencing" reaction set up differently than a regular
PCR reaction? - ANSWER-1. only one primers instead of 2
Complete Answers
When is Sanger Sequencing typically used? - ANSWER-It is not used for routine
bacterial diagnosis. It is only used for new/emerging diseases.
What is the typical run length? - ANSWER-500-700 bp
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
A micoarray is testing for: - ANSWER-a lot of genetic characteristics from one
sample
Southern Hybridization - ANSWER-extract DNA
probe w/ DNA
Northern Hybridization - ANSWER-extract RNA
probe w/ DNA
Western Hybridization - ANSWER-extract proteins
probe with antibodies
What is the first method that became standard in DNA sequencing? - ANSWER-
Sanger method
What type of PCR does Sanger Method use? - ANSWER-cycle sequencing PCR
Cycle Sequencing PCR requirements - ANSWER-1. template DNA
2. buffer
3. 1 primer (drives the region we sequence)
4. dNTPs and ddNTPs (the label)
5. Taq DNA Polymerase
Sanger procedure; current method - ANSWER-1. DNA extraction
2. PCR run
3. Take product and and put on sequencing machine
4. computer generates data
What is another name for the Sanger method? - ANSWER-Dideoxy- chain
termination
In what two ways is this "cycle sequencing" reaction set up differently than a regular
PCR reaction? - ANSWER-1. only one primers instead of 2
