Molecular Methods Flashcards
Usage of methods of molecular biology
Discovery of new genes & proteins Study of gene regulation & protein function Evolution study Diagnosis of pathogens Diagnosis of genetic diseases Production of medicaments Food industry Gene engineering Forensic medicine Criminalistics Identification of animals Pedigree tests
Usage of methods of molecular biology - evolution study
Phylogenetic maps
Usage of methods of molecular biology - diagnosis of genetic diseases
DNA mutations
Usage of methods of molecular biology - production of medicaments
Vaccine, insulin
Usage of methods of molecular biology - food industry
Identification of ingredients
Usage of methods of molecular biology - gene engineering
production of GMO
Usage of methods of molecular biology - forensic medicine
paternity & maternity tests
Usage of methods of molecular biology- criminalistics
Identification of persons
Usage of methods of molecular biology - identification of animals
Illegal tranport
DNA Isolation material
Virus,bacteria, parasites, plants, animals
Body fluids
Tissues, hairs, feather
Fresh/frozen/in parafin
Cloning
Formation of new copies (clone)
Cloning types
Molecular
Embryonal
Transnuclear
Cloning - molecular
Copying DNA by PCR or copying in bacteria
Cloning - embryonal
Division of embryonal cells that results in identical twins
Cloning - transnuclear
Transfer of nucleus from somatic cell into sex cell results in forming the copy of individuum
PCR PRinciple
Forming of identical copy of DNA
PCR Prepare Mixture
Isolated DNA Synthetic primers Nucleotides Taq polymerase Buffer with Mg2+ ions
Gel electrophoresis principle
Separation of DNA fragments according to their size
Gel electrophoresis procedure
Prepare agarose gel (add ethidium bromide)
Stain sample (w/ bromphenol blue)
Lay sample into gel, set electrophoresis
Visualize DNA fragments under UV transluminator
DNA Cloning in bacteria
Small circular DNA with:
Replication origin
Place for restriction endonuclease
Gene for some specific characteristic (antibiotic resistance)
RFLP procedure
Cleavage of DNA by restriction endonuclease
Analysis of DNA fragments on agarose gel
Usage of RFLP
Paternity & maternity tests
Identification of individuum
Identification of bird sex
Studyy of polymorphism & relationship
Southern blotting principle
Transfer of ssDNA from gel into nylon membrane by equipment “vacuum blotter” under vacuum
Northern blotting
Analysis of RNA
Western blotting
Analysis of proteins
Hybridisation principle
Specific DNA probe is bound to ssDNA based on complementarity
DNA probe
ssDNA
Marked radiactive or non-radioactive
Hybridisation procedure
Nylon membrane with blotted DNA is closed into plastic bag together with marked DNA probe
Hybridisation of probe
Removing of non-bound probe from nylon membrane
Detection - autoradiography or luminescence
DNA Sequencing principle
Setting precise order of nucleotides in DNA in equipment “sequenator”
Methods of sequencing
Maxam-Gilbert sequencing
Sanger sequencing
Sanger sequencing
Faster, simpler, no use of toxic substances
Determines the nucleotide order of a given DNA fragment
Sanger sequencing method
Uses same principle as PCR, when template DNA is amplified by help of primers & polymerase
During sequencing, just 1 primer is used & that is why just 1 DNA strand is synthesised in 1 direction
Sequencing reaction takes place in 4 tubes (4 reactions)
Result is mixture of DNA fragments of different size starting with primer & ending with certain dideoxynucleotide
DNA fragments can be separated based on its size by gel electrophoresis & set nucleotide order
Sanger sequencing ingredients
Template DNA Primer DNA polymerase Standard deoxynucleotides 1 of the 4 dideoxynucleotide triphosphates
Dideoxynucleotide
Modified nucleotide
Lacking a 3’-OH group required for the formation of phosphodiester bond between 2 nucleotides. thus terminating DNA strand extension & resulting in DNA fragments of varying lengths
Automatic sequence
Variant of enzymatic DNA sequencing
Dideoxynucleotides are marked with different fluorescent colors (4 diff. colors)
DNA synthesis takes place in 1 reaction
Capilar electrophoresis is used to separate DNA fragments based on their size, fluorescent color is detected by detector at the end of capilar & thus set the nucleotide order
Application of DNA sequencing
Research Medicine Phylogenesis Anthropology Criminalistic Forensic science Agriculture
DNA sequencing - research
Sequencing of whole genome or just part of genome (study of evolution)
DNA sequencing - medicine
Diagnosis of genetic diseases & predispositions (cancer, cardiovascular diseases), gene therapy
DNA sequencing - phylogenesis
Study of evolution
DNA sequencing - Anthropology
DNA comparison
to find out migration of human king (mitochondrial
DNA, Y-chromosomal DNA)
DNA sequencing - sequencing
Using of repetitive sequences of DNA (microsatellites, minisatellites)
DNA sequencing - forensic medicine
Paternity test
DNA sequencing - agriculture
GMO
GMO
Organism whose genetic material was altered using genetic engineering techniques (recombinant DNA technology)
What is DNA microchip?
Small glass plate with 1000s of short synthetic ssDNA (for diff. genes)sequences on the surface
What is a DNA microchip used for?
Research tool used to identify mutations in genes that can cause disease
Researchers will produce chips to help assess individual risks for developing different cancers, heart diseases, diabetes, etc.
How does a DNA microchip work?
Obtain DNA from patient’s blood & control DNA without mutation in monitored genes
Denature DNA, cut into small fragments &label them by fluorescent dye
Both sets of labeled DNA are inserted into chip & allowed to hybridise to the synthetic monitored genes on the chip
Do twins have identical DNA?
Identical twins (monozygotic)
Formed when 1 fertilised egg splits
Identical DNA
Different phenotype - result of interaction between genes & environment in the uterus
DNA test can’t determine the difference while fingerprint can
Chimera in greek myhtology
Monstrous creature that combined the parts of goat, lion, serpent
Chimera in genetics
Animal that has 2 or more different populations of genetically distinct cells that originated in different zygotes
Human chimeras - blood chimeras
People with more than 1 blood type
Human chimeras - identical twins
Embryos share blood supply in placenta, allowing blood stem cells to pass from one and settle in bone marrow of other
Human chimeras - non-identical twins
Blood cells come from twin that died in early gestation
