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Dr: A. Ellethy

College of Dentistry

Lec. 2

1 4 3 1

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What, why, when, where study gene?

How to design a primer?

Types of practical used primers

How to read DNA sequences (the most recently

developed program in USA).

Data analysis for sequenced DNA

Top secretes to study DNA sequences

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What,

why,

when,

where

study

gene?

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WHERE IS DNA? WHY?

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Under sterile conditions

Automated DNA sequencer

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PCR MIX Volume

H2O 12 ul

5X Q soln 5

10X buffer 2.5

50X dNTPs 0.5

primer A 1

primer B 1

Taqpolymerase 0.5

To amplify DNA sequences?

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Labeled ddNTPs with

Radioisotopes S35

Fluorescentlylabeled

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1 cycle: 97C 2 min

30 cycles:

Denaturation 97C 1 min

Annealing XC 1 minExtension 72C 1min

1 cycle:

72

C 6min

store:

4C store

X based melting temperature: 55-60 C

PCR PROGRAME

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A G C T

Start to read DNA sequences

HOW TO READ RADIOGRAPHIC SHEETS OF DNASEQUENCES?

2 hours run 1

4 hours run 2

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TGCTTATTACTGCA

GTCACCATC

-------------------?

A G C T

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DNACHROMAS

Open and save sequences in Applied Biosystems, Staden Chromatogram (SCF), FASTA,EMBL, GenBank, SwissProt, GenPept, GCG RSF and plain text formats.

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HOW TO USE DNA CHROMAS?

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Cell Chromosome DNACell Chromosome DNA

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DNA SEQUENCE

ANALYSIS

IDENTIFICATION

CONFIRMATION

APPLICATION

DIAGNOSIS

VACCINATION

REPAIRING

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ATGGTCTTATTAAGAAAAAACTTCTGTTGTACCATAGGTTATGCCCATATTTCCTCCACCACAACTTCAA

ATCCTTCATCAGAAACGAAGAGCATAGCCACAGACTATGAAACCACCCCGACCACCACTATATCTAGCAC

TGCCTCAGGATCTACGCCCACCCTGACCACCACTGCATCCAGCATTGCCTCAGACTCTAAGCCCACCCTG

ACCACCACTGCATCCAGCACTGCCTCAGGATCTATGTCCACCCCGACCACCCCTGCATCCAGCACTGCCT

CAGGCTCTACACCCACAACGACCACCACTGCATCCAGCACTGCCTCAGGATCTACACCTACCCCGACCAC

TACTGCATCTAGCAAAGCCTCAAGATCTGTGCCCACCACTGTATCCAGCACTGGCTCAGGCTCTACACCC

ACTCCGACCACCACTGCATCCAGCACTGCCTCAGGATCTACGCCCACCCTGACCACCACTCCATCCAGCA

CTGCCTCAGGATCTACACCTACCCCGACCACCCCTGCATCCAGTACTGCCTCAAGCTCTTCACCCACCCC

AACCACCCCTGTATCCAGCACTGCCTCAGGATCTACACCCACCCCGACCACCACTGCATCTAGCAAGGCC

TCAGGATCTGTGCCCACCACTGTATCCAGCACTGGCTCAGGCTCTACACCCACTCTGACCACCACTGTAT

CCAGCACTGTCTCAGACTCTACACCCACCCCGACCACCACTGCATCCAGCACTGCCTCAGGATCAGCACC

CACCCCGACCACCACTGCATCTAGCACTGCCTCAGGATCTATGCCCACCCTGACCACCAATGCATCCAGC

Dentin Sialophosphoprotein EST

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Put original DNA sequences

Put original test sequences

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Blastn dentin sequences

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)1430-1431(

150 50

.

100 .