genetics Flashcards
(32 cards)
what is Molecular genetic methods in medicine
used for?
Manufacture of medicinal products
Detection of pathogens
Identity and related issues (court medicine)
Diagnostic questions:
Diagnosis of monogenic and hereditary diseases
The investigation of genetic risk for diseases
Preconceptive / Preimplantation / Prenatal Diagnostics
Pharmacogenetics
Gene therapy
how to diagnose Genetic diagnosis of Fragile-X syndrome
Cytogenetic examination: cell culture on folate-poor substrate „break point”
(not used since ~1990) due to:
lot of work;
expensive;
takes long;
high rate of false positive and false negative results
PCR-based examination
(for screening only)
Southern-blot-based examination
(most appropriate)
The basic principles - the most important molecular genetic methods can be classified into four types
“Cut-and-paste”–> Restriction endonucleases + ligases
Visualize Electrophoresis
Hybridization–> E.g. FISH
Amplify—> PCR
1) Cut and paste: The restriction endonucleases
application?
restriction endonucleases- enzymes that cut DNA at specific (palindromic) recognition nucleotide sequences
blunt and sticky ends.
application- recombinant dna techniques;
1) restriction endonulcease,
2) sticky ends are sealed together with dna ligase-> recomb dna.
used for gene cloning and inserted into vector (bacteriophage
other application of dna recombination
proteins hormones, vaccine- antigens, antibody, cytokines growth factors coag factors etc.
features of expression vectors
Gene for expression does not
contain intron
ORF codon is optimized
May contain Tags helping in
protein purification
Optimal enhancers, promoters
and teminators
Replication origin
Contain selection
2) Separation and visualization ofDNA / RNA fragments: gel electrophoresis
Agarose concentration in the gel ~ size of the pores (the more concentrated, the smaller)
The negatively charged nucleic acid molecules are drawn through the gel matrix by means of an electric field.
The smaller molecules can move faster through the gel Separation of the fragments according to their size
Stained by eg. Ethidium bromide + UV light - not specific
Stained by eg. Ethidium bromide + UV light - not specific
Many samples at the same time
Main advantage is that different gel matrix can be used – different separation (RNA, DNA, protein)
Special advantage in the separation of DNA sequences (eg. PCR products) of very small size (1bp) difference
3) Hybridization
The basis of genetic methods: DNA-RNA structure A variety of modern methods are based on the ability of double helix formation of nucleotide chains.
hybridization of the complementary strands
DNA-DNA, DNA-RNA, RNA-RNA binding
steps of southern blot
dna 32p labled size markers,
2) electrophoresis and migration
3) solution passes through gel and filter to papertowel.
4) dna transferred to filter.
5) hybridize /w 32p labeled nucleic acid probe.
6) wash away unwanted probe.
7) x ray filter the hybridized complementary sequence.
Applications of Southern blot
Detection of Fragile X syndrome
Identification of methylation sites
in both cases methylation sensitive
restriction enzymes are used
Formerly– to detect point mutations, SNP-s
DNA fingerprint
Comparison of Blot-Hybridization techniques
southern- dna, uses labled dna /w restriction digestion and the result is restriction map.
northern identifies rna, with the use of a dna probe, NO restriction digestion and detects amount and size of RNA.
western blot detects protiens, uses a antibody probe! no restriction digestion and.
4) Amplify: PCR and its variants
Heat stable DNA polymerase and reverse transcriptase enzymes allow PCR and RT-PCR
Polymerase chain reaction-PCR
generation of thousands to millions of copies (amplification) of a or a few copies of DNA sequences in a short time (about 2 hours) (in vitro DNA synthesis)
The thermostable DNA polymerase (Taq polymerase) enzyme of Thermus aquaticus (lives in extremely high temperature) is needed
Necessary: three temperature dependent steps- melt anneal and extend- repeat cycle.
Applications of recombinant DNA technology
polymorphisms in human genome (~ 5%) - may be used for markers
~ 65 millions SNP, 30 thousands microsatellite (STR,
VNTR) are known
- recognition site of restriction enzymes or the distance between them may change due to polymorphisms and mutations (SNP, STR or VNTR InDel)
Size of fragments may be checked by electrophoresis, different cleavage results different fragment pattern = RFLP (restriction fragment length polymorphism)
Southern - RFLP
Genetic diagnosis with RFLP analysis and detection with Southern blot
(RFLP: restriction fragment length polymorphism)
The probe used in the Southern blot hybridizes to a portion of the phenylalanine hydroxylase (PAH) gene that is responsible for phenylketonuria, and detects a restriction fragment length polymorphism with two alleles
PCR-RFLP
1) The DNA region is amplified with specific primers in PCR
2) Digestion of DNA with restriction enzymes (for PCR-RFLP).
3) Gelelectrophoresis to identify of cleavage pattern (genotype)
4) Staining with DNA-binding dyes, eg. Ethidium bromide
5) Evaluation in UV light
VNTR-marker
VNTR - (variable-number tandem repeats) may be in coding, or non-coding regions
Between two individuals restriction sites show variations in length
VNTR show unique patterns: DNA fingerprint
VNTR can be
minisatellite- 10-60bp,
microsatellite- 1-4bp
(STR-short tandem repeat)
VNTR is inherited codominantly
PCR-VNTR
1) The DNA region is amplified with specific primers in PCR
2) Digestion of DNA with restriction enzymes (for PCR-RFLP)
3) Gel electrophoresis
4) Staining with DNA-binding dyes, eg. Ethidium bromide
5) Evaluation in UV light
clinical significance of vntr
1) Triplet repeat diseases (TRD) – expansion of triplet (eg. CAG) in mutant allele
Wild type allele - few triplet repeat (below a limit), but in mutant the number of repeats are multiplied (above a limit)
blood groups
deletions
Multiplex PCR
Allows simultaneous amplification of multiple target regions within single reaction using different primer pairs.
PCR products have to be of different length
Simultaneous detection of several pathogenic E.coli strains found in dairy products
Sequencing
Determination of the nucleotide sequence of DNA fragments
Maxam-Gilber and Sanger methods
Sanger method (chain termination) is widespread
Automation with labelled di-deoxynucleotides and capillary electrophoresis
Detection of point mutations: sequencingSanger method
Chain termination synthesis
1) PCR reaction with dNTP and ddNTP (dideoxyribonucleotides)
2) Because of the absence of 3’OH, the synthesis ends when ddNTP is incorporated
3) Shorter and longer fragments arise
Capillary
4) electrophoresis: all four ddNTPs fluorescently labeled (four colors)
5( Laser reading: colored ladder peaks base sequence
The sequence of nucleotides and the corresponding signal strength is analyzed.
Color-coded chromatograms facilitate evaluation.
on the chromatogram only one nucleotide specific signal is shown in homozygotes (for a nucleotide).
on the chromatogram two nucleotides specific signals (which are a little decreased) can be seen in heterozygotes
Genotyping by sequencing
Aim: not looking for a specific known mutation or polymorphism, but for sequence differences in a not too large, known gene
700 -1000 bp can be sequenced
Detectable: point mutations, smaller insertions, deletions, duplications, heterozygosity, mitochondrial heteroplasmy
Applications of sequencing:
clinical suspicion of cystic fibrosis, detection of hundreds of mutations,
Mutation detection in dystrophin gene, galactosemia (GALT), galactokinase deficiency (GALK1), neurofibromatosis (NF-1)
mitochondrial DNA analysis
In case of infection outbreak sequence analysis of viruses and detection of propagation
