DNA Recombiant
Summary points:
*Because restriction enzymes can digest DNA from any source, DNA from different species can be pieced together *Joining of DNA fragments treated with the same restriction enzyme is done by the action of DNA ligase *Plasmids are extrachromosomal circular elements in bacteria, carrying antibiotic resistance genes *By selection for antibiotic resistance, plasmid DNA can be amplified 1000-fold. *By plating out bacteria harboring a plasmid with an insert the inserts can be purified from the bacterial colonies. *Inserts are characterized by restriction mapping or DNA sequencing *The same principles apply for bacteriophages, bacterial artificial chromosomes and yeast artificial chromosomes except for the amplification *Characterized inserts may then be cloned in another vector (subcloning) for expression.
Summary Points Continued
*In specialized cDNA libraries the cDNA insert is fused to another gene fragment so the function of the protein coded for by the cDNA insert can be studied. *Gene fragment codes for GFP protein: for localization studies *Epitope for antibody: for protein isolation *Yeast two-hybrid for protein-protein interactions *Stable expression of a foreign gene is obtained by integration of the gene construct in the host genome *Transgenic mice can be made by microinjection of gene constructs in the pronucleus of fertilized eggs *miRNAs and siRNAs are key elements in regulation of gene expression by degradation of mRNA's or inhibition of translation *Engineered siRNA represent a versatile tool to modify gene expression
Summary points
*PCR is both a tool to analyze the DNA as well a tool to clone DNA *For PCR a heat-stable DNA polymerase is required *PCR can amplify DNA up to 10,000 basepairs *For PCR-amplification of mRNA or viral RNA, the mRNA has to be reverse transcribed to DNA first *The ratio of Dideoxy nucleotides to Deoxy nucleotides determines the length of the DNA fragments in DNA sequencing *In dideoxy sequening the DNA sequence are read 5' to 3' from the bottom to the top of the sequencing gel *PCR and DNA sequencing products are usually identified by measuring the fluorescence of a fluorescent group bound to a primer or bound to the dideoxynucleotide
Describe the Sanger Method
- 1 of 4 DIdeoxynucleotides is added to a tube containing all 4 normal deoxynucleotides, DNA poly, primer & template strand - DIdeoxy. competes with the corresponding normal nucleotide for insertion into strand -Once added, next nucleotide cant add and polymerization is terminated -Repeated with each of the 4 DIdeoxynucleotides -Gel electropheses (5"->3' read bottom to top)
summary of RNAi
1) A double stranded mRNA (either endogenous or exogenous) is processed by Dicer - Creation of targeting sequence 2) One strand of the processed double stranded mRNA is then incorporated into RISC - Integration of targeting sequence into the missile 3) Argonaute (slicer) attaches to the complex - Addition of nuclear warhead 4) Now either there is a good match between targeting sequence and target which causes a breakdown of the mRNA (boom)... or There is a not so good match and it simply inhibits translation (smaller boom)
Restriction site length determines fragment length
4 bp restriction site: gives fragments of 256 bp on average (4^4) 6 bp restriction site: gives fragments of 4096 bp on average (4^6) 8 bp restriction site: gives fragments of 65536 bp on average (4^8)
Restriction Map
A restriction map represents a linear sequence of the sites at which particular restriction enzymes find their targets
What are the names for formation of Base pairs with their complementary strands?
Annealing or Hybridization
If host cells are bacteria what are some common vectors and what are their libraries?
Bacteriophages (cDNA) Plasmids (cDNA) ---------- Bacterial Artifical chromosome (genomic) Yeast Artificial chromosome (genomic)
What is ChIP? function?
Chromatin immunoprecipitation to find regulatory DNA sequences on a global scale i.e look for transcription factors bound to DNA Antibody binds to the transcription factor and then get "immunoprecipitated" (turns solid so it can be isolated)
How was CF discovered
Chromosome walking
What is cDNA
DNA composed from mRNA via a Reverse Transcriptase
Steps in a typical cloning experiment
DNA sample cut with a restriction enzyme Vector cut with same enzyme at a specific sequence (the cloning site) Restricted vector and sample DNA are mixed and allowed to anneal The annealed overhangs are sealed by DNA ligase. The sample DNA incorporated in the vector we call insert DNA ligated DNA mixture is transfected into bacteria Subsequently the bacteria are plated out DNA library prepared
What are the steps in identifying a specific sequence of DNA?
DNA transferred to solid support (nitrocellulose paper) DNA denatured to single strand (via Alkaline solution) SS DNA hybridized with probe Base pairing of DNA with probe-DNA identified on a blot
What do you know about restriction enzymes?
Endonucleases Very specific, recognize 4-6 Bp of DNA sequence DNA Sequence = Palindrome Produce "sticky" single stranded ends/tails
Remember: LIGASE MUST BE USED EVERYTIME YOU INSERT INTO A VECTOR
FOR BOTH cDNA and DNA library
Bare minimum facts to remember for PCR
For PCR you need *Thermostable DNA polymerase (Taq) *dNTPS (all 4) *Template DNA **2 primers *Buffer solution/Cations(Mg, etc.) **Know it proceeds in cycles - Denaturation, annealing, elongation - bare minimum **Can amplify sequence up to 10,000bp
DNA polymerase of PCR must be _____
Heat-Stable b/c DS DNA is denatured every cycle
IPTG
IPTG is an lactose analog, and by binding to the repressor it prevents it to bind to the operator site Inducer
Which of the following sets of primers would be used to amplify the CAG repeat in the brackets? CAGTCCCTCAAGTCCTTC[CAG] nCAACAGCCGCCACCGCCG
Identify what is "upstream" or 5'.. Assume it's whatever is to the left if the sequence is unlabelled. The upstream stuff IS your first primer Your second primer is the COMPLEMENT of the downstream primer, but make sure you write it 5'->3' CAGTCCCTCAAGTCCTTC & CGGCGGTGGCGGCTGTTG
Differentiate between "Foreign DNA" and "Carrier DNA"
In DNA Cloning (used to amplify DNA) "Foreign" = Dna you want amplified Carrier = vector
Stable gene expression in cultured animal cells
Integration in host genome Neo gene codes for G-418 resistance (NEO) Only works if integrated in host genome
Diagnostic Test for Sickle Cell Mutation
Introducing restriction site or removing restriction site?
What are the steps involved in PCR?
Isolate segment of DNA wanted to be amplified Heat to separate strands Cool to add oligonucleotide primers for synthesis of DNA Add heat-stable DNA Polymerase Heat and Cool again now that Primers & Polymerase present Repeat for 20 cycles In matter of minutes, you have over a million-fold increase
What is the location and length of PCR primers?
Location of primer is just beyond the ends of the segment to be amplified Any 20 Bp sequence will do
How to prepare a cDNA library
Lyse cells from a specific tissue and purify mRNA Hybridize mRNA with a poly (T) primer Make DNA copy with RT Degrade RNA with RNase H Synthesize a complementary DNA strand using DNA polymerase; RNA fragment acts as primer DS-cDNA copy of original mRNA
Real-Time PCR
More rna in red than blue, therefore mRNA is at higher concentration in red
Transient gene expression in cultured animal cells
No integration into host genome Need viral origin of rep for plasmid proteins to be expressed in eukaryotic cells
Subcloning of a DNA sequence for expression
Please note: Shine-Dalgarno sequence needed in vector if using bacterial cells
Whats the difference between polyacrylamide nad agarose gels? How are both visualized?
Polyacrylamide - separate smaller size DNA that differ in length by only one nucleotide Agarose gels: separate longer fragments with larger size differences Ethidium bromide used to visually stain
What is PCR and its purpose?
Polymerase Chain Reaction in vitro method used for rapid production of very large amounts of specific segments of DNA used for clinical or forensic testing since only small amount of DNA required (single drop of blood, semen, hair, etc)
RNAi (RNA interference)
RNAi is a mechanism that inhibits gene expression (silencing) by destroying mRNA or inhibiting translation. The gene silencing is mediated by small RNA s of 20-30 nucleotides in length, derived from longer double stranded RNA precursors by a Rnase called Dicer Dicer is the enzyme that processes ~ 75 nucleotides long genome encoded RNAs with profound hairpin structures into double stranded RNAs (siRNAs) of ~ 23 nucleotides in length. Dicer does the same for added double stranded RNA
What is another name for "Chimera" and how does it come about?
Recombinant DNA Same endonuclease cleaves both foreign and carrier DNA This produces complementary "sticky ends" Foreign DNA added to vector, Base pairs form between comp. sequences and DNA ligase binds together to form Chimera
How to make recombinant DNA
Replication: pUCori is the origin of replication Selection: ApR gene serving as a selectable marker G148 used for selection Expression: NeoR is a bacterial gene it requires eukaryotic promoter sequence (SV40 enhancer) to be expressed and a polyadenylation signal (SV40 pA) Purification using the myc epitope (Ignore f1 ori)
Bare minimum facts to remember for sequencing
Same basic deal as PCR except: *You also need ddNTPs - you need all four, if you are doing it by hand you would need four different reaction vessels. If automated, know you can use one reaction vessel because the four ddNTPs are each labelled with a different colored dye *You would only use one primer (usually) *Can only sequence up to about 1,000 bp at a time *KNOW WHAT IS MEANT BY "TEMPLATE"
The most common procedure for determining the sequence of nucleotides in DNA strand was developed by who? and what does it use?
Sanger Dideoxynucleotides
Yeast Two Hybrid System
Screens for interacting proteins Protein of interest is "bait", fused to DNA binding domain Proteins that bind to "bait" are "fish", fused to transactivator domain (activate transcription for HIS receptor gene)
In order to detect a specific sequence, DNA is transferred to a solid support...what is an example of this type of "solid support"?
Sheet of Nitrocellulose Paper
What is a probe?
Single Stranded polynucleotide of DNA or RNA used to ID a complementary sequence on a larger SS-DNA or SS-RNA Must carry a label in order for identification (i.e. radioactive or flourescence)
SNoW DRoP....what does this mean to you?
Southern blot: DNA hybridized with DNA Probe Northern blot: RNA hybridized with DNA Probe Western blot: Proteins separated, with Antibody probe
ZOO blot analysis to determine interspecies DNA
The KARP-1 LR DNA is apparently primate specific The Ku86 DNA is evolutionary strongly conserved Note that restriction fragment size differ between species Look for bright bands
Northern Blot Gel Interpretation
The amount of strands under the "nuclear" column indicated that this mRNA is still undergoing post-transcriptional modification prior to being transported to the cytoplasm (Specifically, splicing is represented)
Which ONE of the following sequences is most likely to be a restriction enzyme recognition sequence? (A) (5') G T C C T G (3') C A G G A C (B) (5') T A C G A T (3') A T G C T A (C) (5') C T G A G (3') G A C T C (D) (5') A T C C T A (3') T A G G A T
The answer is C. C follows a palindromic sequence of CTNAG, where N can be any base. None of the other sequences is this close to a palindrome. Although most restriction enzymes recognize a "perfect" palindrome, where the sequence of bases in each strand are the same, others can have intervening bases between the regions of identity, as in this question
What is an epitope? what is its purpose?
Where an antibody binds to fusion with peptide to which an specific antibody is available in order to isolate the insert cDNA encoded protein or fuse
Forensics
Which Male was the father of this child? line up lines (male 1)
Reverse Transcriptase produced DNA uses ______ as its template. Because of this template what is a key difference compared to that of restriction enzyme DNA?
mRNA used as template produce cDNA of the RNA DNA produced contains no introns due to the fact that the mRNA template
Use of pBR322 (plasmid) to clone Foreign DNA
pBR322 cleaved at Amp-R by PstI (restriction enzyme) Foreign DNA also cleaved by PstI cleaved pBR322 ligated to foreign DNA *(Note amp resistance is disturbed, not Tet resistance) Transformation of E.Coli Cells on agar containing TET Individual colonies transferred to plates for testing (i.e: one plate with TET, one plate with TET +AMP) So if it grows on the amp+tetra plate then the plasmid was not properly inserted (no foreign DNA present)
Production of insulin in humans
produced from precursor "proinsulin" Bacteria are unable to process human proinsulin, therefore both chains are separately produced in bacteria
siRNA vs miRNA
siRNAs (short interfering RNAs) are Dicer products of exogenous long double stranded RNA precursors miRNA's (micro RNA;s) are Dicer products of genome-encoded long double stranded RNA precursors (endogenous)
What is Gel Electrophoresis?
uses electrical field to separate molecules on basis of size
