Biotechnology and Synthetic biology

what is PCR?

polymerase chain reaction

• allows DNA replication in vitro, multiplying segments of target DNA up to a billionfold during amplification (doubling)

how do we calculate how many copies can be made per cycle of PCR?

2^N copies

• N = number of cycles

• typically around 30 cycles

  • 2^ 30 cycles

  • hypothetically 1 Billion

what is req for the mechanism of PCR?

requirements:

• template DNA

• thermocycler

  • cycles thru diff temperatures

• DNA primers

• ligase

• thermostable DNA polymerase (copies DNA)

  • taq polymerase from T. aquaticus

• dNTPs

  • monomers of DNA

what is the general mechanism of PCR?

each cycle

• melt DNA

  • ~95C

• anneal primers

  • ~55C

• DNA pol extension

  • ~72C

what do the DNA primers do in PCR?

1. specify target sequence to be amplified

2. give an end for DNA pol to add to dNTPs to

3. can encode restriction sites

what drug does Kary Mullis credits his coming up with PCR to?

LSD

who is tom brock and what did he do?

Tom Brock vacation in Yellowstone

• intrigues by hot springs

• isolates first extreme thermophiles > 80C

• thermus aquaticus

  • capable of growth at 85 C

  • how do they survive? → not completely known

  • DNA polymerase in T. aquaticus was found to be extremely heat stable, surviving temperatures of 95C and copying DNA at 72 C

• Emeritus professor of Bacteriology at the University of Wisconsin - Madison

in what ways can we apply PCR?

cloning, sequencing, phylogenetic studies, amplifying very small DNA quantities, medial diagnostics, forensic science, etc.

how does sequence targeting occur by the Cas9 protein?

• Cas9 proteins of CRISPR systems function as endonucleases when guided to nucleic acids

• synthetic RNA (synthetic guide RNA [sgRNa]) that recruits Streptococcus Cas9 and binds to target DNA enables cutting in genome of almost any cell; DNA can be ligated or used to insert new DNA

• homologous recombination can be used to incorporate new researcher designed edit with a repair template

how do we create indels with CRISPR?

to create an “indel”, rather than incorporate specific researcher designed edit, non-homologous end joining is used w/o a repair template

how can we achieve molecular cloning?

recombinant DNA: combining sequences from multiple sources to create novel DNA

what does recombinant DNA technology depend upon?

the ability to produce large numbers of identical DNA molecules (clones)

how are clones generally generated?

by placing a DNA fragment of interest into a vector (plasmid) DNA molecules, which can replicate in a host cell

when a single vector containing a single DNA fragment is introduced into a host cell, large numbers of fragments are reproduced along with the vector

an overview of gene cloning (3 steps):

1. isolation and fragmentation of source DNA

   • can be amplified by polymerase chain reaction, synthesized by reverse transcriptase, or synthetic DNA made in vitro

2. insertion of DNA fragment into cloning vector

   • small, independently replicating genetic elements that can carry and replicate cloned DNA

   • designed to allow insertion of foreign DNA at a restriction site

3. introduction of cloned DNA into host organism

what are cloning vectors?

• several types

• use dependent on purpose

• plasmids widely used

  • contains resistance gene

  • contains multiple cloning site

    • stretch of DNA w unique restriction sites

  • contains origin

    • specific # of copies of plasmid that are around

what is the difference between a restriction enzyme and Cas9?

RE: only cuts at a very specific spot in the DNA

Cas9: can cut anywhere so long as guide RNA brings it to that spot

what are restriction endonucleases and how do they work?

DNA-cutting enzymes

• each enzyme recognizes one or a few target sequences and cuts DNA at or near those sequences

why doesn’t RE cut host DNA?

The enzymes are often designed to recognize and cut foreign DNA, such as that from viruses, while ignoring their own modified DNA sequences.

what is left of the strands after REs cut them?

they make staggering cuts, producing ends with single stranded DNA overhangs

• some produce blunt ends

what is one of the major roles of REs?

protect against viral infection

how does DNA ligase work?

joins/anneals DNA

• if source and vector have complementary sticky ends from being cut by the same restriction enzyme, DNA ligase can anneal them

• if source DNA is PCR generated, primers can contain extra DNA encoding restriction site

What bond is cleaved by the restriction enzymes?

phosphodiester bond between 5’ PO4 and 3’ OH

steps to cloning:

slide 20

what is then done with this recombinant DNA?

transformed into suitable host for replication → this is the actual cloning process

what is sanger sequencing?

1. PCR with fluorescent, chain-terminating ddNTPs

2. size separation by capillary gel electrophoresis

3. laser excitation and detection by sequencing machine

what are the steps to gibson assembly (another type of DNA sequencing)?

1. PCR of dsDNA fragments with overlapping ends

2. 5’ exonuclease chews back one end of each of the dsDNA fragments, producing ssDNA overlaps

3. DNA fragments anneal

4. DNA polymerase extends 3’ ends, replacing the DNA chewed back by the 5’ exonuclease

5. DNA ligase seals nicked DNA

why is Gibson assembly convenient?

• don’t need RE or restriction sites

• uses overlapping fragments and allows us to stitch two pieces of DNA together

• one reaction with all these enzymes

what is nucleic acid hybridization?

complementary base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix

• segments of known single-stranded DNA used in hybridization are nucleic acid probes

• detection occurs with radioactivity or colored/fluorescent products

what is the purpose of nucleic acid hybridization?

can be used to determine whether or not a particular gene is being expressed

• ex: gene probe

  • not expressed in planktonic growth

  • expressed in biofilm growth

what is FISH?

Fluorescent In Situ Hybridization: uses fluorescent probes to target specific sequences in cells

how is FISH useful in the medical field?

identifies pathogens in clinical samples or bacteria in environmental samples

what is reporter gene fusion?

coding sequence from reporter is fused with regulatory region from another source

• can use to study gene expression

• first widely used was lacZ from E. coli encoding beta-galactosidase

• Green fluorescent protein (GFP) common

what is gel electrophoresis?

employs an agarose gel to separate nucleic acids by size and charge

• nucleic acids migrate thru gel toward the positive electrode because of their negatively charged phosphate groups

• small molecules move faster than large

compare with ladder (standard sample)

not finished go back and look at the last 4 slides in the presentation