Lecture #48

• the number and morphology of nuclear chromosome are detected and analyzed.
• done during the metaphase phase of mitosis
• any cell type

• phosphate backbone is stained with stain Giemsa
• during metaphase

• largest to smallest
• MSA ( metacentris, submetacenstric, acrocentric)
• p are up and q arm down

• sex chromsome last
• chromsome 22 is bigger than 21

• Aneuploidy
• specific chromsomal anomalies
• deletions with various sizes in given chromsomes

• detect aneuploidy 
• specif anomalies 
• deletions

• from larges to larges
• Metacentric, submetacentric to acrocentric

Diving cells

• G-banding with trypsin and Giemsa
• the dark staining shows heterochromatin 
• the light stain show euchromatin

the number of bands that can be seen is increased

• 450-550 band resolution 
• detects mega base changes ( >5-10 Mb)

Tumor biopsies, bone marrow, peripheral blood.

resolution btwn 550-850Mb due to cells being arrested in prometaphase and prophase instead because chromsomes are less dense at this time

• unexplained multiple congenital anomalies
• mental retardation
• developmental delay 
• dysmorphic features

• it detects and localize
• Flourescence In situ Hybridization 
• detection and localication of specific sequence
• evry pari of chromosome produce two dots

• e it relies solely on chromosomal morphology for
• identification of an abnormality. This limitation can be overcome with fluorescent in-situ
• hybridization (FISH), which specifically binds to target genetic sequences

designing a flourescent dye probe ( aka probe tagged with fluorophore) with a complementary sequence to detect a target complementary DNA sequence.

When the target Dna anneals with the Dna of interest in the body. A then counter stain can be added when the sample is taken on a slide and  you will have to use a flourescent microscope

• interphase does not have much seperation of chromosome so you can see chromosomal regions
• does not need dividing cells
• use locus-specific probes and satelittle dna probes

• also called M-FISH Multiflex FISH 
• each of the 24 chromosome get a sorting probe. 
• each chromosome is labeled with their own color flourochrome
• The chromosome are mixed all together then karyotyping 
• you can see translocation

an individual chromsome in a batch of chromsome that is labeled with it own distinct color of using flurochrome

• complete karyotyping with automated analysis
• origin of marker chromosome, small insertions and complex rearrangement i.e. translocations

they are chromosomal regions

• resolution 2Mb
• better than karyotyping because of resolution 
• show you dividing metaphas cells
• multiple probes can be used
• detects bacteria
• detect microdeletions/microdupliations, aneuploidy, and derivative( rearranged) chromosomes

• high resolution <1Mb
• rapid
• can use paraffin embedded section 
• you can make prenatal diagnosis fo frequent numeric chromosomal anomalies using amniocentresis or oral mucosal swab

Trisomy 21, 18, 13and x/y alterations

• you take a patients genome and you take reference control DNA
• when they are mixed you add them onto the micro array and what happens is an hybridization that gives off a specific color depending on how many copies are sustained after mixing
• yellow mean there was no change
• red there was a lost of copies and green there are more copies( a duplication )

only relative copy numbres are known and it need to be confirmed by FISH or KAyotyping

• scanning the entire genome and comparing a patients genome to a  reference sample.
• you can align and see what specific changes have happened

• 2 types
• increasing the amount of genetic material. through molecular cloning and PCR

• it is a circular piece of DNA use in duplicating a a specific gene.
• it can replicate autonomously in a host cell

• extra DNA, chop them up with restriction enzymes ( this cut at specific DNa sequences). Then you use a library and find a cloning vectors to insert this DNa intpo the vector. 
•  you then put the vector in s a bacterial cell and they will multiply this specific gene.

• you can use this to detect mutations.
• DNa is amplified in a living system

• Advantages
• • Within one day
• • Detects specific mutations in many samples simultaneously
• Disadvantages
• • Impractical for large no. of different mutations
• • Some enzymes are expensive
• • Only limited no. of point mutations affect restriction sites

• polymerase chain reaction
• amplifies Dna exponentially
• relied on DNA polymerase  DNA primers along with specific cycling conditions

• cleave dna with restriction enzymes that 
• take Dna fragments and insert into plasmids using DNA ligase

• lyse cells
• etxra mrna
• hydrolyze with polt T primer
• make DNA copy using reverse transcritase 
• Rnas to get rid of most RNA complementary to newly formed DNA
• use DNa polymerase to create a complementary Dna strand 
• clone into vector 

• There are three main enzymes.  are isolating mrna in this library 
• reverse transcriptase can crea dna from the mrna in the cdna library.
• RNase 
• Dna polymerase

• isolate a genomic Dna
• find specific primers that whill bind to the single srand of that Dna 
• add everything in a a tube with nucleotide. 
• and DNa polymerase

detect HIV

with all the necessary component in the test tube, you then raise the temperature to separate the dna

• Technique to separate charged samples of interest according to SIZE 
• small sizes migrate faster while the larger sizes take longer and linger more toward the negative starting end.

• - Both procedures begin with isolation of RNA followed by characterization for purity and
• integrity. Purified RNA is then used as template to generate first strand cDNA.
•  Both endpoint PCR and real-time PCR data
• analysis require normalization of data to known standards to determine relative or absolute
• quantity of starting target gene expression.

During endpointPCR, DNA is measured at the completion of PCR amplification. Quantification of DNA product isdetermined by gel electrophoresis, staining of separated DNA fragments with a fluorescent dye,and digital imaging densitometry to measure DNA band intensity. In real-time PCR, DNA ismeasured during the exponential phase of PCR amplification. Accumulating product is detectedas it is being amplified using fluorescent DNA probes.

• RTPCR
• continos flourescent measurement of PCr product during each cycle of PCR
• EPPCR
• Dna gel electrophoresis of PCR product
• quantification of visible PCR products by densitometry