Lecture 11 Flashcards
1
Q
How do you isolate RNA?
A
- in eukaryotes: mRNA’s end in a stretch of A’s called the Poly-A tail
- use poly-T to base pair with poly A tails
1. wash off and left with all mRNA (not a singular particular one)
2. wash RNA and collect rest
2
Q
what are restriction endonucleases (RE)/ restricition enzyme?
ex. EcoR1
A
- cut dsDNA at specific sequences called restriction sites; recognize 4-6 bp sequences and cut
- naturally occuring
- some can create sticky ends that have overhangs
- in E. coli strain RY13, the endonuclease EcoR1 finds the restiction site (palindrome sequence; same seq on both strands)
- cuts dsDNA with a staggered cut
- unpaired bases can base pair with any DNA that shares same overhangs/ sticky ends
- RE don’t cut their own host DNA becuz: bacterial DNA is methylated to hide any genomic restriction sites
3
Q
What is Gel Electrophoresis?
A
- separation of DNA based on size
- DNA is negatively charge therefor is attracted to the positive charge in an electric field
1. load DNA sample into cells at top
2. apply an electronegative current such that negatively charged DNA is attracted to the (+) end
3. compare to the ladder to approximate fragment length
4. to visualize: need to add a chemical that intercalated into DNA and glows under UV light
4
Q
Finding sequences in a gel
what is the basic blot technique
A
- goal is to find a unique seq in a mixture separated on a gel
- use of a probe: a single stranded DNA seq labelled with a fluor (a fluorescent molecule)
- probe anneals to the complementary sequence and expose the fluor to create a band on film
5
Q
What is a Southern Blot?
what are you doing and what is it good for?
A
- looking for a specific DNA seq on a gel
- detect presence of a seq of interest
- compare known genes from a known genome to an unknown genome
- must denature dsDNA into ssDNA to allow probe to anneal
6
Q
What is a Northen Blot?
what are you doing and what is it good for?
A
- run RNA gel
- RNA’s represented will be gene actively expressed
- do no need to denature RNA on gel b/c already ssRNA
- use ssDNA probe
- determining gene expression patterns
- band length can indicate splicing patterns
- band thickness is informative on expression levels
7
Q
What is RFLP (restriction fragment length polymorphisms) mapping
A
- using a highly polymorphic locus that does not have exolutionary pressures to identify individuals
- fingerprint mapping
need: - RE
- DNA Gel
steps:
1. cut isolated DNA with specific RE
2. run on gel
3. probe with ssDNA probe sepcific to known RFLPS - loss or gain of RE site will change pattern on gel
look at notes
8
Q
What is GWAS (genome wide association studies) and explain it
A
- SNPs = single nucleotide polymorphisms
- SNP map in the human genome has been determined with up to 700,00 diff SNPs
- assess linkage of SNPs to known or unknown genetic loci that maybe controlling a trait
- ex. human height
- used to find what genes are involved and how many are they
- compare with Manhattan plots
9
Q
Define Cloning
what are the tools for cloning?
A
- replicate a piece of DNA in a foreign host
- produces a lot of gene of interest
- dependent upon the ability of DNA to anneal to any other DNA as long as the sticky ends are compatible
- adhering if the ends are complementary
tools - RE
- plasmid vectors (carry DNA to and from)
- ligase - glue DNA fragments together
10
Q
What are plasmids?
A
- small independent replicating pieves of circular DNA extra to the chromosome in prokaryotic cells
- they can be manipulated/ genetically engineered to carry DNA from other species
- plasmids designed for clonding are called vectors
11
Q
Explain vectors
pUC19
A
- have been largely engineered to contain an MCS - multiple cloning site, a site for positive and/or negative selction
- pUC19 has lacZ gene
parts
1. MCS - RE cut MCS to linearize the vector
2. ori = origi of replication seq; bacterial seq that allows replication of vector in E.coli
3. ampR gene= confers antibiotic (ampicillin) resistance; allows selection for successful transformation b/c only bacteria with plasmid will survive
4. selectable marker (Lac Z); select for successful cloning
12
Q
what are the steps to cloning?
A
- prepare DNA insert; RE digest and cut out specific band from gel
- prepare vector; cut vector with same RE for compatible sticky ends
- Mix and ligate with ligase; base paired but sugar phosphate backbone not so ligase joins SPB of insert to vector
- Transform into competent cells; take vector and insert to E coli.; there will be undesired combos but look at which colonies successfully took up the right vector
- select for succesful transformants; grow on amp, grow on X-gal and see if colony is white or blue
- confirm; linearized or 2x to separate insert and vector
13
Q
Sources of insert DNA
Explain cDNA (complementary DNA)
A
- reverse transcribed mRNA
- expressed genes; processed mRNA’s therefore no introns/regulatory regions
- cDNA library = set of bactiera that each contain a cloned DNA
- it can be a snapshot of gene expression
14
Q
Sources of insert DNA
What is PCR ( polymerase chain rxn)
purpose and tools
A
- amplify seq of interest from a mixture
tools: - need a template; need seq information in deatil to design artifical primers
- need 2 primers (15-20ntds); ssDNA sequences; complementary and must sit on either side of the seq you want to amplifiy and have their 3’ ends pointing in; must anneal to complementary strands of DNA
- need themostable DNA polyermase; Taq and Pfu
- dNTPS
- buffer which includes Mg (essential cofactor for pol)
15
Q
What is the process of PCR
A
- take seq of interest and denature it for 2 mins at 95oC
- annealing; putting primers in for 1 min at 55oC
- polyermization; copy of DNA template running length <4kb at 72 oC for 1 min
- repeat `25-30x (95-55-70)
- dominant product in the tube DNA b/w and including primers
