Basic Tools Of Recombinant Dna

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DNA isolation steps

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Cloning

Sequencing

Access function

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Name restriction enzynes

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Initials of genus (1st letter)

Species (2nd & 3rd letter)

Strain (4th letter)

If multiple restriction enzymes have been isolated from same organism then designated sequentially by Roman numeral

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Steps in DNA cloning

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Cut open plasmid and paste in gene ( restriction enzymes& DNA ligase)

Transform plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up plasmid

Grow many of plasmid carrying bacteria use them as factories to make the protein

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RE are highly specific and recofnize specific sequences (most palindromic). The longer the recognition sequence the _____

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Less frequent the number of cuts

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RE cut sites can be

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Blunt end enzyme cut straight through

Staggered produce sticky ends that can anneal to complement (such as on a plasmid) & ligase can join those ends = RECOMBINANT DNA!!

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Digestion using RE produce fragments and they cut with specific frequencies based on what factors?

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Base composition

Frequency of sequence

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Restriction Modification System

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RE pairs with metylase

Enzymatically catalyze the addition of methyl group

RE recognize specific UNMETYLATED DNA

*This system prevents RE from cutting up host DNA since prokaryotes lack compartmenralization

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Compare contrast agarose & polyacrylamide gel

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agarose less resolving ability larger range for molecule size

polyacrylamide gel more resolving ability and narrow range

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EcoR1

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Produce sticky ends

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Sma1

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Produces blunt ends cut straight down middle

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RE fxn in bacterial cell

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Prevent invasion by foreign DNA By cutting it into fragments

Cut at sites within foreign DNA instead of catalyzing from the ends

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Restriction Modification System

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Enzymes recognize and methylate the same site

Methylation protects DNA ,after replication the parental strand is already metylated

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The greater the concentration of agarose the

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Smaller the pores

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SYBR

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Real time PCR & nucleic acid staining

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Visualization of bands use DNA binding dyes give example

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Ethidium bromide

SYBR

Thiazin (fast blast & methylene blue)

Nile blue sulfate

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Significance of restriction maps?

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Possible to determine the regions of biological importance in circular viral DNA

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Transducing bacteriophage

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Naturally occurring vectors that carry parts of bacterial chromosomes from one bacterial cell to another

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Much larger fragments can be closed in _ than in plasmids

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Bacteriophage

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All plasmids are

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Easily purified from E coli

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pUC plasmids

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40% of DNA has been deleted

Cloning sites clustered together in one area (multiple cloning sites)

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Benefits of MCS

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Allows one to cut vector & foreign gene with 2 different restriction enzymes

Allows use of directional cloning technique to know the orientation of the insert

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Plasmid insert range

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<10 kb

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Phage insert range

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<23

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P1 artificial chromosomes (PAC) insert range

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130-150kb

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Cosmid insert range

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30-46

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Bacterial artificial chromosomes

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<300kb

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Yeast artificial chromosomes insert range

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200-2000

Eukaryote!!!

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5 basic steps of cloning

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Choose appropriate DNA (genomic or complementary DNA)

Make fragments of suitable size to fit into appropriate vector carrying resistant gene

Insert DNA fragments into vector and use DNA ligase to link the DNA fragment to vector

Vectors with inserted DNA are introduced to bacterial population (TRANSFORMATION)

transformed bacteria are plated on agar with antibiotic

Only bacteria with plasmid will grow they are then plated at low density so that each bacterial cell that divides gives rise to a colony

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Complementary DNA

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DNA copy of mRNA made using reverse transcriptase

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2 common screening methods for plasmids ?

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Antibiotic resistance allows for selection of bacteria that received copy of vector

Blue/white selection multiple cloning sites inserted into lac z (code for enzyme B-gal

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When do we use cDNA (RNA - DNA hybrid)

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When we want to understand protein fxn

Interested in a.a sequence

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When do we use genomic DNA

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Regions of gene that regulate gene expression

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Isolate mRNA from total cellular DNA (mostly rRNA & tRNA)

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Use oligo dT attached to beads that will attach to the mRNA polyA tail (post transcriptional modification ) in eukaryotes

MRNA can be eluted from column

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How do you prepare DNA copy of RNA for cDNA library

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Use oligo dTs to act as primers for reverse transcriptase

RT uses RNA to make DNA strand

Product rna DNA hybrid

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Convert rna DNA hybrid into ds DNA for cloning

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Use RNaseH enzyme that recognizes hybrid molecules and digests RNA fragments

Fragments serve as primers for DNA Polymerase

Ligase joins nicked DNA

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Discuss two ways to make sticky when making cDNA Library

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Use RE

Terminal transferase enzyme to add poly-C tail to 3' end of vector and then

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dNTPs

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Deoxy nucleoside triphosphates

Nucleotides (A,T,C,G) attached to 3 phosphates

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Southern blotting

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Combination of gel electrophoresis with hybridization (DNA Sequences called probes that are complementary to sequence of interest) useful in detecting sequences

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Southern blotting made possible the

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Production of detailed restriction maps.

Used in conjunction with electrophoresis creating maps covering hundreds of kilobases

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Northern blotting

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RNA sample electrophoresed blotted, hybridized with labled probe

Determine which tissues express which genes, gene regulation

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Western blot

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Detects protein with antibodies

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Fluorescence in situ hybridization

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Uses fluorescent probes to bind to complementary parts of chromosome. Detects presence or absence of specific DNA sequences

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DNA Sequencing

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Fredrick Sanger

Sanger dideoxy Method

2',3'Dideoxynucleosides (ddNTPs) of each base are made

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Reverse transcriptase must start at the 3' end of mRNA (PolyA tail) as a result RT may not reach the 5'end of the molecule how is this fixed

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Oligo fragments are made up of many possible sequences in this way priming of DNA occurs from many positions

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Polymerase chain reaction

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Allows production of a lot of specified DNA without having to clone.

Used DNA Polymerase to make multiple copies of target sequence of DNA

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Optimization of PCR product depends on

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Temperature (primer & DNA Polymerase )

Salt concentrations

Primer design (forward &reverse)

Annealing temperature

G/C content ( multiple H bonds hard to break)

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Pfu polymerase has

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Proofreading capabilities

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Reverse transcriptase PCR (RT-PCR)

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When using RT-PCR in cDNA cloning :

Start with mRNA not ds DNA

began by converting mRNA to DNA

began by converting mRNA to DNA Use forward primers to convert ssDNA to ds DNAContinue with standard PCR

forward primers to convert ssDNA to ds DNA

Use forward primers to convert ssDNA to ds DNAContinue with standard PCR

Continue with standard PCR

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Pfu polymerase has

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3 to 5 exonuclease activity.

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2 sticky ends permits directional cloning in

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RT PCR

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qPCR (Real time PCR)

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Quantifies application of DNA as it occurs

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During qPCR As the DNA strands separate they anneal ____

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Forward & reverse primers

Fluorescent tag oligonucleotides complementary to part of one DNA strand that serves as reporter probe

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In qPCR what serves as the reporter probe

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Fluorescent tag oligonucleotide serves as reporter probe. Probe had fluorescent tag @ 5' end and a quencher 3' end

Fluorescence increases with incorporation into DNA and can be quantified

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How does the quencher fxn in qPCR

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When quencher is within certain distance of probe the quencher stops flourescence

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As PCR progresses ___ & ____ are broken apart and separated by DNA Polymerase which stopes ____ allowing flourescence to occur

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The probe (5) and quencher (3)

Flourescence

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Derailed gene analysis involves the following steps

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Isolate gene by cloning

Be able to manipulate sequence

Be able to return the gene to cells/organism to determine fxn