Molecular Techniques in Human Genetics

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Molecular Techniques in

Medical Genetics

By: Babak Nami


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Contents:

Molecular cloning

Restriction enzymes

VectorsPlasmid

Genomic libraries

cDNA libraries

Screening of library

Southern blotting

Allele specific oligonucleotide

Northern blotting (RNA blotting)

PCR

qPCR

Study of DNA sequences

FISHCGH

RNA Expression Arrays

Western blotting of proteins


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Molecular Cloning


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Restriction Enzymes


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Vectors

"Vector" is an agent that can carry a DNA fragment into a hostcell. If it is used for reproducing the DNA fragment, it is called a

"cloning vector

Types of vectors:

Plasmid: Have a capacity of 15 kb.

Phage(lambda)s: Have a capacity of 25 kb.

Cosmids or Fosmids: Have a capacity of 35-45 kb.

Bacterial artificial chromosomes(BAC)(P-1 derived): Have a capacity of 50-

300 kb.Yeast artificial chromosomess(YAC): Have a capacity of 300- >1500 kb.

Human artificial chromosomes(HAC): Have a capacity of >2000 kb.


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Plasmids


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Phage


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Cosmid


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YAC


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Genomic Libraries

A genomic library is a population of host bacteria, eachof which carries a DNA molecule that was inserted into

a cloning vector, such that the collection of cloned DNA

molecules represents the entire genome of the source

organism. This term also represents the collection of allof the vector molecules, each carrying a piece of the

chromosomal DNA of the organism, prior to the

insertion of these molecules into the host cells.


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cDNA libraries

A cDNA library is a combination of cloned cDNA (complementary DNA)fragments inserted into a collection of host cells, which together constitute

some portion of the transcriptome of the organism.

cDNA is reverse transcription from RNA. It comes in very handy when

working with mRNA. mRNA contains introns and exons, and during post

transcriptional modifications the introns are spliced out. You only have exons

present now, which is different than that made from the genomic DNA. This is

what makes up about 20,000 different proteins in Humans. Also accounts for

most of the enzymes present in any cell type. The advantage is, that you can

have a library that encodes directly the proteins in a cell, and that way you can

know the proteins makeup. Also it would make it much easier to makeproteins for cellular analysis


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Hybridization and Probes


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Screening of libraries


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Southern blotting


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Northern blotting


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PCR


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Sanger sequencing


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FISH


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CGH


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array-CGH of a glioblastoma


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Western blotting of proteins

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Molecular Techniques in Human Genetics