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ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑ ΡΑΜΠΙΑΣ ΘΕΟΔΩΡΟΣ. ΕΦΑΡΜΟΣΜΕΝΗ ΜΟΡΙΑΚΗ ΒΙΟΛΟΓΙΑ. - PowerPoint PPT Presentation

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DNA, RNA , DNA DNA (Sanger Sequencing)BLAST DNA cDNA (qPCR, qRT-PCR) (Microarrays)Deep sequencing

(PCR) DNA

PCR: 1985, Nobel Kary Mullis in 1993

+ MgCl2+ Buffer + dNTPs+ Taq polymerase+ (primers) DNA

, UV ! ! (PCR) DNA ) )

PCR :Magnesium chloride: .5-2.5mM

Buffer: pH 8.3-8.8

dNTPs: 20-200M

Primers: 0.1-0.5M

DNA Polymerase: 1-2.5 units

Target DNA: 110 g/ml 9

(PRIMERS) : 20-30 GC = 40-6-% .

104 DNA PCR 25-30 ... 110 g/ml DNA.

DNA +

m Wallace formula: Tm = 4x (#C+#G) + 2x (#A+#T) C

gCl 2 Taq polymerase!!!

PCR

PCR

PCR Mg++ (annealing)

MgCl2 PCR.

MgCl2 1 mM 3mM

Stock of primers: 100pmol/l :

50pmol/l, 25pmol/l, 12,5pmol/l,6,25pmol/l 3,125pmol/l .

..19

42-57,5C

..20

, DNA

DNA 1/2

Cloning and selection markers

Antibiotics

The problem of self ligation

-Need for additional selection markers

Generation of engineered restriction sites on plasmids on selectionmarkers

LacZ, Blue-white selection

+INSERT

LacZ, Blue-white selection

Applications of PCRBasic ResearchApplied ResearchDetection of pathogensPre-natal diagnosisCancer research-Mutation detectionMolecular Epidemiology

GenotypingGenomic cloning Sequencing+Real time PCR+cDNA synthesis and quantitative RT-PCR

Basic Research_Molecular cloning

DNA DNA . DNA. DNA ( short tandem repeats STR) , , DNA, 1-6 bp multiplex PCR, DNA . (pattern) (unique barcode) .

37For identification purposes, the unique pattern of DNA fragments generated by amplifying variable DNA regions is not useful by itself. The unique pattern must be compared to another pattern to determine if the patterns match. Matching patterns indicate that the two DNA samples originate from the same individual. Short tandem repeat analysis examines repetitive regions of DNA where a single, short DNA sequence is tandemly repeated multiple times. Single-nucleotide polymorphism analysis examines differences in nucleotide sequence at a single position within the DNA. STR and SNP analysis can be useful in any investigations where DNA evidence can be collectedfor example, a blood stain or fingerprint from a crime scene or a bone in a missing persons investigation. DNA extracted from these samples is amplified to generate a unique set of DNA fragments. DNA is also extracted and amplified from a reference sample (blood samples taken from a suspect while in police custody or the missing persons toothbrush or hair brush). The DNA pattern from an unknown source (e.g., DNA found at a crime scene) is compared to the DNA pattern from the reference sample. Matching patterns indicate that the sources of the DNA are identical. STR and SNP analysis can also be used to determine paternity. Since a child inherits half of his or her DNA from the father, half of the childs DNA fragments will match those of the biological father.

Short tandem repeat

DNA FRAGMENT ANALYSISSTR1STR2STR3STR4

DNA-Based Human Identification

40In this example, the STR loci D16S539, D7S820 and D13S317 were amplified by PCR, and the resulting DNA fragments were separated by size. The numbers to the right of the gel image indicate the number of times that the DNA sequence is repeated (5 repeats is denoted as allele 5; 10 repeats is denoted as allele 10, etc.,). Lanes marked L indicate the allelic ladder, which contains DNA fragments corresponding to known STR alleles (alleles 515 for D16S539, alleles 614 for D7S820 and 715 for D13S317) . By comparing the sizes of DNA fragments in the allelic ladder to those of an unknown sample, a scientist can determine the number of repeats in the unknown sample. If you look closely, you will see that none of the DNA patterns for samples #16 match. Thus, all six DNA samples originated from separate individuals, and none of the five suspects can be linked to the hair based on this DNA evidence.

DNA detection of pathogens

DNA .. PCR

PCR

41Because of its speed and sensitivity, PCR is often the preferred method to detect DNA sequences that are not normally present in a particular sample. One example is detecting bacterial or viral DNA in human blood to aid in the diagnosis of infections and disease and help guide treatment decisions. Also, PCR can be used to detect potentially harmful organisms in our water or food supply. RT-PCR can be used to detect specific RNA sequences, such as viral RNA.PCR can be instrumental to detect specific DNA sequences when identifying and characterizing an organism. For example, the multidrug resistance (MDR) gene product confers resistance to antibiotics, and treatment of an infection differs if the organism is MDR+ or MDR. PCR is often used to characterize an infectious agent and its virulence factors to guide the course of treatment.PRC diagnosticsViruses HIV, SARS, H5N1Bacteria meningococcus, legionellosisAnalysis for resistant genes MRSA, VRERT-PCR can detect specific RNA sequences within a sample.Example: Retroviruses have an RNA genome. Retroviral RNA can be detected by RT-PCR to diagnose retroviral infections.

Detection Of Pathogens

Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)Molecular Identification:Mutation detection

Genomic PCR amplification

Sanger Sequencing

Sanger sequencing

PCR_

cDNA synthesisRECOMBINANT PROTEINS

Production of recombinant InsulinT7 RNA Polymeraseis anRNA polymerasefrom theT7bacteriophagethat catalyzes the formation ofRNAin the 5' 3' direction.

pET System