DNA Technology Flashcards
Cytogenetics
Cytogenetics is the branch of genetics that studies the number and morphology of chromosomes, and there relationship to disease.
Karyotype
is the complete set of all chromosomes of a cell of any living organism
Methods/characteristics of cytogenetics
1.Culture blood in media and bovine calf serum
2.PHA - a mitogen (a peptide or small protein that induces a cell to begin cell
division) is added to stimulate white blood cells to divide in culture
3.Centrifuge cells - white blood cells go to the bottom of the tube (red cells on
top)
4.Hypotonic solution - causes white blood cells to swell and ruptures the red
cells
5.Fixation of cells - stabilizes cells and chromosomes
6.Drop cells onto slides - cells burst open and chromosomes fall out
7.Band chromosomes by partially digesting the chromosome with trypsin and
staining with Giemsa
8.Analyze under the microscope or imaged
Chromosomal numberical abnormalities
• Aneuploidy
• Extra chromosomes – usually lethal
• 21 Downs, 47,XY,+21
• 18 Edwards
• 13 Patau
• Missing Chromosomes
• Turner syndrome 45X
- Additional sex chromosome
Chromosomal rearrangements
Deletion
Duplication
Inversion
Insertion
Translocation
Restriction Fragment Length Polymorphism (RFLP)
• Restriction enzymes generally originate from bacteria and sometimes archaea, and appear to be used as a defence against invading viruses
• Restriction sites are mostly palindromic.
• Some restriction enzymes produce blunt ends some sticky overhanging ends. These sticky ends can be useful for cloning and other methods needing DNA ligation.
Gel electrophoresis
-Mixture of DNA molecules of different sizes
-negatively charged dna molecules will move toward the positive electrode
-Shorter molecules are slowed down less than
longer ones, so they move faster through the gel.
DNA profiling pros and cons
+Large amount of DNA required
-DNA must be high quality
+Time: A few days
-Sensitive to large scale contamination
DNA finger printing pros and cons
+ Can be done using only a few cells
+ Works with degraded DNA
+ Time: A few hours
-Very sensitive to contamination
PCR
Polymerase chain reaction (PCR) is a method for the exponential amplification of small sections of DNA. The ability to replicate specific sections of DNA revolutionised the field of genetics.
• PCR uses a pair of primers specific for the sequence to be amplified
• PCR amplification occasionally incorporates errors into the amplified
strands and so cannot substitute for gene cloning in cells
• You can use different polymerases. For example, in addition to 5 ́to 3 ́ DNA polymerase activity Pfu polymerase, it also possesses 3 ́ to 5 ́ exonuclease (proofreading) activity, so it has a lower error rate than Taq.
Human genome project
• Human reference genome
• International collaboration
-using the Sanger method
Sanger sequencing
-Millions of copies of the section of the DNA to be sequenced are produced using standard PCR.
-A sequencing reaction is then carried out using a single primer and including a radioladled ddNTP. The reaction had to be run for each of the dNTPs.
-This sequencing reaction produces different sized products depending when the ddNTP terminates the sequence. Gel electrophoresis allowed the fragments to be separated by size
Bacterial artificial chromosome
Chromosome fragments are created with overhanging sequences at either end that will match those in the bacterial plasmid.
A gene that makes bacterial cells resistant to an antibiotic is present on the plasmid.
As the bacteria is grown in medium containing the antibiotic only cells that take up a plasmid will survive.
Bacteria can be used to accurately replicate larger sections of foreign DNA than could be produced using Sanger sequencing.
The bacteria is plated out thinly on an agar plate so that each single cell grows into a separate colony. These colonies can be grown to supply large quantities of the plasmid.
Primer walking
Primers designed in the known bacterial sequence of the BAC are used to start the sequencing process but the BAC is to big to be sequenced in one go so new primers are designed and the primers are ‘walked’ together.
Shotgun sequencing
1 Cut the DNA into overlapping fragments short enough for sequencing.
2 Clone the fragments in plasmid or other vectors.
3 Sequence each fragment.
4 Order the sequences into one overall sequence with computer software.
