anaylzing cell systems 2

Question 1

Some facts to remember

Answer 1

1/1000 nuceoltudes are different b/w human genomes

1.5% of DNA is exonic DNA

Single nucleotide polymorphisms (SNP) can be neutral, pathogeneic, or predisposing

Question 2

What are restiction endonucleases?

Answer 2

Bacterial Enzymes that cut DNA in specfic sites can produce clean cuts or overhangs

Question 3

What is agarose gel electrophoresis ?

Answer 3

Different than SDS-PAGE

DNA is already charged,

smaller dna seperated faster than larger

Question 4

What is a ligase reaction?

Answer 4

Using Ligase to glue DNA strands back together (requires ATP)

Question 5

Bacterial plasmids are used to do what?

Answer 5

1. Used as cloning vectors
2. Cleavage of segment of DNA via restriction nuclease
   
   1. endo nuclease cuts same strand
   2. restriction makes overhang but cutting through both
3. Dna fragment to cloned is inserted (covalent linkage)
4. recombinant DNA

plasmids engineered to serve various functions

Every gene can be put into bacteria and stored indefinitely and propagated at anytime

Question 6

What are cDNA clones?

Answer 6

• DNA copy of mRNAs
• No introns
• Much smaller than original genome
• requires viral enzyme
  
  • reverse trancriptase

Question 7

What is the difference between cDNA and a Genome?

Answer 7

Genomic DNA = exons and introns

cDNA = Exonic

Question 8

What is “simple description” PCR?

Answer 8

Polymerase Chain Reactions

Amplifies DNA regions

1. heats DNA to seperate strands
2. cool to anneal primers (additon of primer here)
3. DNA synthesis

Process is repeated until the job is done

Question 9

What is the “complex” def for PCR?

Answer 9

• DS dna obtained from patient, individual, or pathogen

1. high temperature to denature or seperate into ssDNA
2. Primers designed to complement sequence that flank each end in 3-5’ direction
   
   1. Cooled and allowed to anneal (bind to DNA)
3. add dNTP

• TAQ polymearse synthesizes copy of DNA by extending primers or both ends. DNA doubles each cycle and becomes greatly amplified
• reactions carried out in thermocycler - has programmable settings (Temp, Number of cycles etc)
• Advantage = very small amount DNA needed
• Disadvantage = need to seq for flanking portions for primer design, error prone, amplification of contaminating DNA

Question 10

What is one example of application of PCR?

Answer 10

Presence HIV DNA that is amplified by PCR and recongized by gel eletrophoresis

Question 11

What is qPCR?

Answer 11

Quantitative PCR

Used to quantify copy number of a specific gene in two or more samples in real time

In addition to primers, this technique includes a probe which fluoresces only a presence of PCR product

Probe usually complementary oligo with a fluorescent tag

Used to detect levels of infectious agent and determine levele of gene expression

Question 12

What is restriction fragment length polymorphism?

Answer 12

when genomes from 2 individuals cleaved with a set of restriction enzymes and the fragments resolved on a gel pattern is different

SNPs occur in recongniton sequences for restriction enzymes

Used in DNA finger printing

used in foresic analysis, paternity testing and disease detections

Question 13

What is one example of RFLP “restriction fragment length polymorphism” ?

Answer 13

• Normal beta globulin allele has 3 Ddel restricition sites
• Patients with sickle cell only have 2 restrictiion sites
• THis occurs because a GAG -> GUG
  which casuses glu-> val (mut)
• Cleavage of genomic DNA followed by Ddel followed by eeltrophoresis and southerning blotting including a B globin resulted in ……
  
  • The dad only has 2 restriction endonucleo sites and the daughter is carrier of the diease

Question 14

What are variable number of tandem repeats?

Answer 14

variation in short tandom repeats (STR) in individuals

regions are isolated from genomic samples by fanking restriction sites or PCR

useful in ID and severity of of disease

Ex: hunting disease, fragile Xs syndrome , and fredrich Ataxia

Question 15

what is a DNA microarray?

Answer 15

Used to determine overall change in gene expression b/w 2 samples

each spot has a different DNA sequence from a different Gene

used to determine difference in gene expression between 2 samples

1. isolated RNA from sample, make cDNA using reverse transcriptase, and adds a fluorescent dye (called probe)
2. add a different fluorescent dye to 2nd sample
3. inserted into microarray where it will bind to its complementary sequence
4. more intense signal, higher degree of binding and highler level of gene expression

Question 16

what is a paraquat?

Answer 16

Compound that creates reactive oxygen species in cells