روشهایی بررسی و تشخیص جهشها در ژنتییک مولکولی DNA

We now know how God wrote the book of life

But do we know how to read the book ?

Genetic Diagnostics Cytogenetic tests FISH Molecular tests

Molecular Diagnostics - Diagnosis of infectious diseases - Genetic identification - Diagnosis of genetic diseases

Genetic Identification - Paternity Testing - Forensics

Diagnosis of genetic diseases - Somatic rearrangements in cancer - Genetic risk factors - Pharmacogenetics - Mutations in monogenic diseases

Types of Mutations Tested Disease Point mutations? Deletions & duplications? Few recurrent mutations? Many unique mutations? Also with point mutations? Whole gene? Some exons? Other mutations?

Most genetic technologies are based on four properties of DNA 1.DNA can be cut at specific sites (motifs) by restriction enzymes 2.Different lengths of DNA can be size-separated by gel electrophoresis 3.A single strand of DNA will stick to its complement (hybridisation) 4.DNA can be copied by a polymerase enzyme DNA sequencing Polymerase chain reaction (PCR)

DNA can be copied by a polymerase enzyme Polymerase chain reaction (PCR) A method for producing large (and therefore analysable) quantities of a specific region of DNA from tiny quantities PCR works by doubling the quantity of the target sequence through repeated cycles of separation and synthesis of DNA strands 1. PCR: Polymerase Chain Reaction

DNA can copied by a polymerase enzyme

PCR can generate 100 billion copies from a single DNA molecule in an afternoon PCR is easy to execute The DNA sample can be pure, or it can be a minute part of an extremely complex mixture of biological materials The DNA may come from – a hospital tissue specimen –a single human hair –a drop of dried blood at the scene of a crime –the tissues of a mummified brain –a 40,000-year-old wooly mammoth frozen in a glacier. In the words of its inventor, Kary Mullis…

DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA DNA polymerase C C C C C C G G G G G G G G G T T T T A T T A A A A A A A A

DNA can copied by a polymerase enzyme

A C T G DNA template Heat resistant DNA polymerase G, A, C, T bases Forward primer Reverse primer A thermal cycler (PCR machine)

DNA can copied by a polymerase enzyme

Modified PCR and non-PCR amplifications techniques ARMS PCR --- point mutations RT-PCR ---- gene expression(qualitative) Gap PCR ---- deletion Real time PCR ---- gene expression (quantitative)

Modern genetic markers: SNPs ARMS: amplification refractory mutation system Clin Biochem Rev (2006) 27: 63–75 Amplification-Refractory mutation system (ARMS) PCR

Allele-specific PCR (ARMS test)

Restriction enzymes cut double-stranded DNA at specific sequences (motifs) E.g. the enzyme Sau3AI cuts at the sequence GATC Most recognition sites are palindromes: e.g. the reverse complement of GATC is GATC Restriction enzymes evolved as defence against foreign DNA DNA can be cut at specific sites (motifs) by an enzyme Sau3AI 2. RFLP: restriction fragment length polymorphism

ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA DNA can be cut at specific sites (motifs) by an enzyme

Sau3AI ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA DNA can be cut at specific sites (motifs) by an enzyme

Sau3AI ACTGTCGATGTCGTCGTCGTAGCTGCT GATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAG CATCGATCGA DNA can be cut at specific sites (motifs) by an enzyme

ACTGTCGATGTCGTCGTCGTAGCTGCT TGACAGCTACAGCAGCAGCATCGACGACTAG GATCGTAGCTAGCT CATCGATCGA ACTGTCGATGTCGTCGTCGTAGCTGCTGA TGACAGCTACAGCAGCAGCATCGACGACT TCGTAGCTAGCT AGCATCGATCGA DNA can be cut at specific sites (motifs) by an enzyme

Different lengths of DNA can be separated by gel electrophoresis DNA is negatively charged and will move through a gel matrix towards a positive electrode Shorter lengths move faster

Different lengths of DNA can be separated by gel electrophoresis DNA is negatively charged and will move through a gel matrix towards a positive electrode Shorter lengths move faster

Different lengths of DNA can be separated by gel electrophoresis Slow: 41 bp ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA Medium: 27 bp ACTGTCGATGTCGTCGTCGTAGCTGCT TGACAGCTACAGCAGCAGCATCGACGACTAG Fast: 10 bp GATCGTAGCTAGCT CATCGATCGA F M S

Different lengths of DNA can be separated by gel electrophoresis Recessive disease allele D is cut by Sma3AI: ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA Healthy H allele is not cut: ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA F M S HHHD DD

Different lengths of DNA can be separated by gel electrophoresis F M S HHHD DD The C282Y mutation, caused by a guanine (G) to Adenine (A) transition, results in the substitution of cysteine by tyrosine in the HFE protein.

A single strand of DNA will stick to its complement ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA 3.DNA Hybridization

A single strand of DNA will stick to its complement 60°C ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

A single strand of DNA will stick to its complement 95°C ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA 3.DNA Hybridization

A single strand of DNA will stick to its complement 60°C ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

A single strand of DNA will stick to its complement a. Southern Bloting

A single strand of DNA will stick to its complement Southern blotting (named after Ed Southern)

A single strand of DNA will stick to its complement Southern blotting (named after Ed Southern)

A single strand of DNA will stick to its complement

Array technology: 1.Microarray …… Gene expression 2. CGH array…Genomic imbalances

DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA DNA polymerase C C C C C C G G G G G G G G G T T T T A T T A A A A A A A A 4. DNA Sequencing

DNA can copied by a polymerase enzyme ACTGT ACTGTCGAT ACTGTCGATGT ACTGTCGATGTCGT ACTGTCGATGTCGTCGT ACTGTCGATGTCGTCGTCGT ACTGTCGATGTCGTCGTCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGAT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT

DNA can copied by a polymerase enzyme ACTGTCGATGTCGT

DNA can copied by a polymerase enzyme ACTGTCGATGT ACTGTCGATG ACTGTCGAT ACTGTCGA ACTGTCG ACTGTC ACTGT Fluorescence Time Fluorescence Time TGTAGCTTGTAGCT T C G A T G T etc

DNA can copied by a polymerase enzyme

Looking into the future