EXAM2_L20_Molecular_Bio_in_Medicine Flashcards
_________ are bacterial enzymes that cut ______DNA into smaller pieces. They cut in a ______specific and ____ specific manner.
What is an example? Where does it cut? what does it cut?
Restriction endonucleases cut DOUBLE STRANDED DNA
SEQUENCE and ORIENTATION SPECIFIC CUTS
ie. ECOR1 cuts between G/A of sequences GAATTC
Cohesive/sticky ends
Blunt ends
sticky ends sometimes have 2-4ntds overhanging
DNA fragments made by SAME restriction enzyme an be joined together again by DNA ligase
Molecular Cloning-
3 parts needed for a vector
covalently binding recombinant DNA into a vector to make Copies
Vector is Circular dsDNA capable of auto replication inside a host cell.
- Multiple Cloning Sites (one or more palindrome seq.)
- Antibiotic resistance
- Origin of Replication (for auto replicate of vector)
3 Steps to creating a recombinant DNA molecule
- Vector cleaved w/ restriction endonuclease
- Cleave foreign DNA with SAME Restriction Enzyme
- Add DNA ligase to join DNA pieces covalently
Product (recombinant molecule) can be put in bacteria to make large amount of copies.
Gel electrophoresis
What does it do?
What gels used?
Separates DNA by its size
-Uses electricity (migrate to positive end)
-Smaller migrate further
AGAROSE- large pores (separates big differences in size)
POLYACRYLAMIDE- (Small pores- separates one base pair difference)
The Sanger Sequencing method: ddNTPs
(Dr. Fred Sanger)
This works because ddNTP’s stop elongation at each A,T,G,C site and at each length.
Small pieces to large sequences will be plotted in the correct nucleotide channel to indicate the nucleotide, and the gel electrophoresis will separate pieces by size and indicates Where that (A,T,G,C) is in the sequence.
This allows us to get a perfect sequence of the template
dideoxyribonucleotides (ddNTPs)
- derivatives of dNTPs without the 3’ OH group
- chain terminates w/o 3’ OH
- Template DNA to be sequenced is added to 4 flasks
- Primer & dNTPs added to each flask equally
- Radiolabeled ddNTP’s added to each flask individually
(ddATP, ddTTP, ddCTP, ddGTP) *one in each flask - DNA polymerase added
- Polyacrylamide electrophoresis run for each flask (A,T,G,C)
- Sequence read for SIZE (bottom to top) to display sequence of the template.
The new sequence is IDENTICAL to the original strand that the template came from
Fluorescent DNA sequencing (automated)
Improved Sanger Method:
One tube One lane,
Each ddNTP labeled with different dye
Each color ID’d w/ laser & computer
PCR
4 components
3 steps
How many molecules per cycle?
1 tube, 4 components
- dsDNA template
- excess of 2 sets of DNA Primers (one for each strand)
- excess dNTPs
- DNApol (taq. poly)- heat stable
- Denature (95C)- separates DNA
- Anneal (55C)- primers bind each template strand
- Elongation-synthesizes w/ DNApol
EXPONENTIAL GROWTH: #molecules= 2^n (n= #of cycles)
RT-PCR
makes DNA from mRNA (NEAT!)
1. mRNA converted to cDNA via reverse transcriptase
(copy of gene w/ EXONS only- lacks introns)
2. cDNA used as template for PCR (amplification)
Blotting & Hybridization What 3 types of blots? What material analyzed for each? What do each detect? What probe used for each?
Detecting Specific DNA, RNA, PROTEIN in a contaminated mixture
1. Southern (DNA)- detects #copies of a gene or to detect specific genes in DNA— radioactive/floresc DNA label
- Northern (RNA)- detects sizes & amounts of mRNA (transcriptional regulation)—radioactive/floresc DNA label
- Western (Protein)-detects amount of Proteins & differences in post-translational modification
(translational regulation)— enzyme-linked antibody
4 General steps of blotting?
- Electrophoresis (separation by size) DNA,RNA,Protein
- Transfer “blot” onto membrane
- bind Labeled probe
- Visualize by autoradiography or chemiluminescence
How do you detect specific nucleic acids?
What is in situ hybridization?
Base pairing between complementary strands of DNA or RNA.
- Denature at high temp
- re-anneal at low temp with hybrid label strands
In situ- detects DNA or RNA sequences in chromosomes or intact cells
DNA Microarrays “DNA Chips”
thousands of DNA sequences on a microchip
- each spot is a gene
1. genotyping (genetic testing) -Uses DNA
2. Gene expression profiling (genomic test) - use mRNA converted to cDNA via RT-PCR
- detects how much mRNA being made
Two types of microarrays?
- Genotyping (genetic test)- Seq of DNA- AMPLICHIP
(what type of protein being made?) - Gene expression profiling (genomic testing)
Type/quantity of mRNA
(how much mRNA is being made?) ie: cDNA microarray
Genetic vs genomic testing
Genotyping (genetic testing) - amplichip
-test sequence of DNA (what type of protein being made)
Gene expression profiling (genomic testing)
- How much mRNA is being made? (use cDNA microarray)
