DNA Sequencing and Gene editing Flashcards
(89 cards)
- the order of nucleotides in the DNA molecule is used in the medical laboratory for a variety of purposes, including ,,,
DNA sequence information
- detecting mutations
- typing microorganisms
- identifying human haplotypes
- designating polymorphisms
Treatment strategies including_ are now selected based
on the results of these techniques
targeted therapies
is the process of determining the precise order of nucleotides within a DNA molecule.
DNA Sequencing
is just a part or portion of the genome of a particular organism or sample.
DNA Sequencing
Direct determination of the nucleotide sequence, or DNA sequencing,
is the most definitive molecular method to identify ________________.
genetic lesions (mutations)
- Direct determination of the order, or sequence, of nucleotides in a
DNA polymer is the most specific and direct method for identifying
genetic lesions (mutations) or polymorphisms, especially when
looking for changes affecting only one or two nucleotides.
Manual Sequencing
Two types of sequencing methods were concurrently developed in
the 1970s:
Maxam–Gilbert sequencing and Sanger sequencing
Classical or Manual
- Maxam-Gilbert chemical sequencing
- Manual Sanger dideoxy chain termination sequencing
Automated – based on Sanger method (dideoxy chain)
- Automated Sanger sequencing
- NGS or Next Generation Sequencing
- Used in whole genome sequencing and in sequencing several samples of DNA simultaneously.
- Uses several platforms or approaches depending on the manufacturer of the machine.
- There are several available platforms nowadays offered by different manufacturers.
Automated – based on Sanger method (dideoxy chain)
Maxam–Gilbert sequencing
is a method of DNA sequencing developed by _ and _ in _
Allan Maxam and Walter Gilbert
- 1976–1977
was the first widely
adopted method for DNA sequencing, and, along with the Sanger dideoxy method.
Maxam–Gilbert sequencing
Method based on chemical modification of DNA and subsequent cleavage at specific nitrogenous bases.
Maxam–Gilbert sequencing
Before the DNA template is cleaved into
fragments, first, it must be radio-labelled
After the procedure, each fragment will have a ___
Maxam–Gilbert sequencing
- phosphorus 32 (P32) radioactive label
_ part of the phosphate group attached
to the 5’ end of each fragment with a base modifier.
- __ are the tubes
-__ are the ones written on the tube
Maxam–Gilbert sequencing
- P32 forms
- Aliquots
- Base modifiers
chain breaks at
G
G+A
T+C
C
BASE MODIFIER REACTION min@25dc
Dimethylsulphate Methylates G 4
Formic acid Protonates Purine 5
Hydrazine Splits pyrimidine ring 8
Hydrazine + salt Splits only c ring 8
- Involves chain breakage at specific nucleotides.
- In this procedure, the template may either be double stranded or
single-stranded. - Replaced by Sanger sequencing because of certain disadvantages
Most notable disadvantages:
1
2
Maxam–Gilbert sequencing
1. Time consuming – not practical for high throughput sequencing
2. Chemicals used are hazardous
Another reagent is needed in order
for the DNA template to be cleaved
at specific sites, and that is adding
_ which is a strong reducing
agent.
* Upon addition, the template would
break at specific nucleotides
10% piperidine
methylates guanine. When you add 10%
piperidine, the template will cleave at guanine.
Dimethyl sulfide (DMS)
– protonates purines and cleaves at either guanine or adenine.
Formic acid
splits pyrimidine rings, cleaving at cytosine or thymine.
Hydrazine
cleave only cytosine rings.
hydrazine + salt
This will result in molecular fragments called a __, which is then separated
using electrophoresis. This is in order to
separate the fragments according to length.
Maxam–Gilbert sequencing
- sequencing ladder
__ from 6% to 20% are used for
sequencing.
* __ and __ are used to monitor
the migration of the fragments.
Polyacrylamide gels
Bromophenol blue and xylene cyanol loading dyes
