Flashcards in genetics Deck (32):
1
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
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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)
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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
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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
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other application of dna recombination
proteins hormones, vaccine- antigens, antibody, cytokines growth factors coag factors etc.
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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
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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
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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
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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
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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.
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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
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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.
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4) Amplify: PCR and its variants
Heat stable DNA polymerase and reverse transcriptase enzymes allow PCR and RT-PCR
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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.
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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NGS-next generation sequencing
Limit of traditional sequencing: approx. 800 nucleotides
Different NGS methods exist, parallel sequencing of millions of DNA
1 run - hundreds Mb or few Gb sequence
Nowdays used more or less for research, for whole genome sequencing, in the future will be used for diagnostics as well (SureSelect Human Kinome Kit)
Future: to decrease the cost and increase the speed of sequencing.
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ngs steps
1) sequence primer hybdridize to the template.
2) incorporation of each dntp is accompanied by the release of pyrophosphate group.
3) ppi generated visible light can be detected and presented in the pyrogram.
4) an enzyme degrades unicorporated dntps. then the next dntp is added.
5) addition of dntps iis performed one at a time, and the pyrogram generated from the visible light show the sequence..
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PCR-RFLP and 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-b inding dyes, eg. Ethidium bromide
5 Evaluation in UV light
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RT-PCR Reverse transcriptase polymerase chain reaction
For the analysis of mRNA expression- BUT: mRNA is not particularly stable we need cDNA from the mRNA
- cDNA can be analyzed using a traditional PCR reaction
Preparation of cDNA
Primers for synthesis:
An oligo dT nucleotide (15-25 deoxythymidine), complementary to the poly A tail of the mRNA, or
Random hexamer oligonucleotides (consisting of six randomly assembled nucleotides)
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Reverse transcription (RT) PCR
The template is mRNA
is reverse transcribed to cDNA
is amplified by PCR (qualification)
Application of real time PCR allows quantification, to study gene expression, too
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The real-time / quantitative PCR(qPCR)
Is a further development of the "traditional" PCR reaction
Is based on the principle of conventional polymerase chain reaction (PCR)
The reaction is followed in real-time
This allows accurate quantification of the amplified DNA by means of fluorescence measurements
Enables both quantitative analysis of DNA (qPCR, e.g., bacterial DNA, human gene amplification, etc.)
As well as the determination of the amount of RNA (qRT-PCR, e.g., measurement of gene expression, viral RNA, etc.)
It allows to detect the PCR product as it is produced (as the fluorescent signal is generated)
Applications
Genotyping (detection of SNP-s)
Qualitative real time PCR (yes/no)
Quantitative real time PCR (Q-PCR) (g or cDNA)
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Real time – PCR
Quantitative PCR - it allows the scientist to actually view the increase in the amount of DNA as it is amplified.
TaqMan® probe detection
probe with a fluorescent reporter at
one end and a quencher of
fluorescence at the opposite end
of the probe. The close proximity
of them prevents detection of
fluorescence
The probe hybridize to the middle
of DNA region to be amplified. In each
cycle these probes are degreded by
DNA polymerase physically separating
the fluorescent reporter from the
quencher, resulting in an increase
in fluorescence.
Fluorescence is detected and
measured in a real-time PCR machine,
and its geometric increase corresponding
to exponential increase of the product is
used to determine the quantification cycle
(Cq) in each reaction.
In positive samples (containing the studied DNA sequence) after some PCR
cycles fluorescence above threshold is measured
In negative samples the fluorescence remains below threshold
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