hybridization techniques

list different hybridization techniques

• southern blot

• northern blot

• western blot

• southwestern blot

• fot / slot blots

• reverse dot blots

• DNA microarrays

• FISH

• CGH

• DNA sequencing

what does the southern blot detect

specific DNA sequences

how is DNA denatured in the Southern Blot

in the gel by an increase in pH

which probe is used for detection in Southern Blot

labeled complementary probe

how does DNA move in the Southern Blot

DNA transferred to a membrane by capillary action with a high salt solution

Southern Blot procedure

1. genomic DNA cut with restriction enzymes

2. DNA electrophoresed

3. gel submerged in an alkaline solution to denature DNA

4. DNA transferred onto nitrocellulose membrane by capillary action

5. membrane mixed with a solution containing labeled probe

6. membrane washed to remove excess, unbound probe

7. membrane developed and visualized

what will the labeled probe do in Southern Blot

hybridize to complementary piece of DNA on gel

how can the produced membrane from Southern Blot be visualized

• radioactive isotopes

• chemiluminescent dyes

• colorimetric techniques

what does the Northern Blot detect

sequences of RNA

how does RNA move in Northern Blot

RNA transferred to membrane by capillary action using a high salt solution

what is used for detection in Northern Blot

labeled complementary probe

what does the Western Blot detect

specific proteins

what gel is used for the western blot

SDS-PAGE = polyacrylamide gel electrophoresis

Western Blot procedure steps

1. protein run on SDS-PAGE

2. protein electrically transferred to membrane

3. membrane incubated with a primary antibody and blocking solution

4. membrane washed and incubated with secondary antibody and blocking solution

5. membrane washed and rinsed with substrate buffer

6. substrate added and developed

what kind of transfer is seen in Western Blot

electro-transfer

what does the Southwestern Blot detect

DNA-binding proteins

what do Dot/ Slot Blots do

quick analysis of RNA and DNA

what are dot / slot blots applied to

• expression analysis

• mutation analysis

• amplification analysis

do dot / slot blots determine the size of the target

NO

what kinds of probes do Reverse Dot blots use

ASO = allele specific oligonucleotide probes

what is the important method for genotyping common human mutations

reverse dot blots

what occurs in reverse dot blots to test allele

hybridization

Reverse Dot blots are important for

genotyping common human mutations

what does “Stringency” describe

the conditions under which hybridization takes place

what increases stringency

salt, heat, and formamide

DNA Microarrays can also be referred to as

DNA Chip

what do DNA Microarrays use , and for what?

hybridization technology to examine gene expression

what are DNA Microarrays used for

• gene expression studies

• disease diagnosis

• pharmacogenetics (drug discovery)

what do DNA Microarrays need special instrumentation for

• generation of micro arrays

• analysis of results

how many genes foes each microarray have

thousands of genes

DNA microarrays allow one to simultaneously monitor what?

gene expression levels in all these genes

what is a DNA microarray / chip

arrangement of DNA sequences on a solid support

FISH stands for

fluorescent in-situ hybridization

what does FISH detect

detect and localize presence or absence of specific DNA sequence or chromosome

what is FISH able to examine

metaphase chromosome spreads as well as interphase (non-dividing cells)

describe how FISH works

hybridization of a fluorescent DNA probe to its complementary DNA in morphologically preserved tissue or cells

CGH stands for

comparative genomic hybridization

what does CGH provide

a map of chromosomal regions that are gained or lost

what can CGH identify

specific location from which the extra material organized

what does CGH do

compares and measures differences in copy number changes between 2 DNA samples (test and control samples)

what does DNA sequencing determine

the order of nucleotides in a DNA molecule

different types of DNA sequencing methods

• Maxam - Gilbert

• Sanger method

• Pyrosequencing

• next generation sequencing

what type of degradation is used in Maxam-Gilbert sequencing

chemical

what kind of chain termination is seen in the Sanger method

dideoxy chain termination

how does Pyrosequencing work

sequence by synthesis

describe next generation sequencing

• sequence single molecules of DNA in real time

• significant computational analysis

• performed on variety of platforms

gene cloning is also known as

recombinant DNA technology

describe the procedure of gene cloning

1. DNA from 2 different species cut with restriction enzymes

2. ligate into a vector

3. introduce into a proper host cell

4. multiply and code for desired production

what are DNA Polymorphism

differences or polymorphisms reside in several places in the genome

types of DNA polymorphisms

• RFLP

• VNTR

• STR

• SNP

• Y-STR haplotypes paternally inherited

• mitochondrial DNA is maternally inherited

what DNA is maternally inherited

mitochondrial

what is paternally inherited

Y-STR haplotypes

RFLP=

restriction fragment length polymorphism

VNTR=

variable number tandem repeats

what polymorphism is also known as minisatellite

VNTR

STR =

short tandem repeats

what polymorphism is also known as

microsatellite

SNP =

single nucleotide polymorphism

SNP can also be referred to as

single nucleotide mutation

what are some SNPs associated with

various phenotype differences with propensity towards disease and drug resistance

how many SNP locations are possible

> 5 million SNP locations

what could the maternal lineage/exclusion of mitochondrial DNA result in

differences in hypervariable regions of mitochondrial genome