DNA notes

DNA vector


Approximate max. insert size
plasmid 8 kb
insertional λ-vectors 10 kb
replacement λ-vectors 23 kb
cosmids 40 kb
Yeast Artificial Chromosomes 1000 kb

I ruin my life already

I think I don't have any degree offer...
they forgot my application already...
i can't blame anyone... i just blame to myself...
very sad...
no one will understand my situation...
I understand my situation too much...
I can't help crying...

oligonucleotide-directed mutagenesis

Site directed mutagenesis (SDM) is a powerful technique where site specific changes in DNA sequence are produced in vitro - for instance to change an amino acid residue into another by changing the codon sequence within the gene sequence.

SDM is also used to 'engineer' commercially important proteins for many different purposes, for example 1. improve stability 2. change specificity 3. reduce toxicity

Viral hemagglutination inhibition test

still has few day to immunology exam...

still has few day to immunology exam...4 3 2 =>1...

very very nerous...

many many notes...

release ....

just thinking of you are reading a story book!!!

relex ... listen some music than study again !!

After the exam I want to swim, play piano, go to gym room to do some exercise with my dad... (may be hiking too)

then meet some old friends and teachers (primary + secondary school friends)...

than find a summer job or jobs ... of course to earn money

for what? to give me parents for XXX ... hohoho top secret...

Don't understand? just guest it... anyway to share my burden of my parents...

relex

Single Nucleotide Polymorphisms involved in Haemophilia

A single point substitution of the "C" at position 6460 of the clotting factor IX gene for a "T" causes haemophilia . This is because this mutation is a nonsynonymous substitution, which means the alteration of the nucleotide affects the encoded amino acid at that position. The "C" lies in the codon "CGA", which encodes for an amino acid arginine (Arg). Changing this sequence to "TGA" alters the meaning of the codon to a terminator in the protein coding sequence. In this case, an individual with this sequence will not make any clotting factor IX. This means that the blood clotting process fails which can cause haemophilia.

other e.g. of SNP=>Coronary Heart Disease and Debrisoquine Metabolism

Making DNA Fingerprints

1) Isolation of DNA.
small amount of tissue, like blood, hair, or skin

2) Cutting
restriction enzymes
e.g. EcoR1, found in bacteria


The DNA pieces are sorted according to size by a sieving technique called electrophoresis.

3) Transfer of DNA to nylon membrance/nitrocellulose membrance

4) Probing.


Uses of DNA Fingerprints

1. Diagnosis of inherited disorders
2. Forensic or criminal
3. Personal identification

RFLPs in Mapping Genetic Diseases

hemoglobin gene from each family member

Hb A is the wild type allele, and Hb S is the allele that codes for the sickling of red blood cells

RFLP's are produced using this polymorphic DNA sequence and the resulting fragments are separated by gel electrophoresis

The wild-type hemoglobin gene, Hb A, appears at 1.15 kb, while the sickled hemoglobin gene, Hb S, appears at 1.35 kb.

A person homozygous for sickle cell anemia (S/S) shows only one RFLP at 1.35 kb, while people heterozygous for this disease (A/S) have RFLP's at 1.35 kb and 1.15 kb.

People who have not inherited this gene (A/A) show one RFLP at 1.15 kb.

Using RFLPs for mapping genetic diseases and for DNA fingerprinting

Restriction Length Fragment Polymorphisms

gene as a marker

1. sickle cell anemia


2. phenylketonuria

obtain a sample of blood, hair root, or other biological sample.

These fragments are generated by cutting genomic DNA with a restriction endonuclease at a particular nucleotide sequence and separating the resulting fragments on a gel by performing a Southern blot

(G^CGC)

These fragments are then run on an agarose gel in separate lanes and the fragments will migrate towards the positive electrode to different degrees based on the molecular weight of each fragment.

The smaller fragments will move farther on the gel than the larger fragments.

The fragments then need to be visualized. This is commonly done by transferring the bands to a nitrocellulose gel and probing for the various DNA sequences contained in the fragments.

Ideally, this probe is 6-10 bp, but in the simplified example above, the probe could be GCG, which would bind to the red CGC sequence contained by all fragments. The probe needs to be able to be visualized, and this can be done by exposing the radioactive probe to x-ray film.

At this point, the fragments of various lengths can be visualized and the sequence differences between these two people can be visualized. Person one will show 3 DNA fragments and Person 2 will show 2 DNA fragments.