Recombinant DNA: - PowerPoint PPT Presentation

1 / 28
About This Presentation
Title:

Recombinant DNA:

Description:

When the sticky ends of two DNA fragments come together and hydrogen bond ... the DNA polymerase FILLED IN the missing portion of each strand making TWO NEW ... – PowerPoint PPT presentation

Number of Views:236
Avg rating:3.0/5.0
Slides: 29
Provided by: jennifer60
Category:

less

Transcript and Presenter's Notes

Title: Recombinant DNA:


1
Chapter 17
  • Recombinant DNA
  • The Tools of the Trade

2
Restriction enzymes
  • Discovered in the 1970s
  • Used by bacteria to break up viral DNA
  • Bacteria DNA is preserved by being methylated
  • Reads in paladromes
  • The DNA fragment produced by resptriction enzymes
    acting on the DNA of a cell are call genomicDNA
    gDNA

3
(No Transcript)
4
(No Transcript)
5
(No Transcript)
6
  • Animation of the ligation process. When the
    sticky ends of two DNA fragments come together
    and hydrogen bond through the A-T and G-C
    pairing, the enzyme LIGASE will form covalent
    bonds between the two fragments.

7
  • Once you have the DNA you want you will want to
    make many copies of it.
  • PCR (Polymerase Chain Reaction) Nobel Prize 1993
  • AMPLIFYING our DNA millions of time.

8
PCR The components are
  • A sample of the TARGET DNA to be copied. In
    theory only a single molecule is needed.
  • A set of short (15 to 40 bases) single stranded
    PRIMERS of DNA, in EXCESS, that will bind to
    complementary regions of the opposing stands of
    the TARGET DNA molecule . These primers BRACKET
    the region of DNA to be amplified.
  • An EXCESS of the 4 nucleotide triphosphates,
    ATP, GTP, CTP, TTP.
  • The enzyme, DNA polymerase.
  • Various buffers and cofactors like magnesium
    ions required by DNA polymerase.

9
  • The final trick was to get the two target DNA
    strands APART (separated) so the primers could
    bind and the DNA polymerase could do its thing
  • Mullis heated his mixture of target DNA, primers
    and triphosphate nucleotides to about 90oC for a
    few minutes to separate the target DNA.
  • then lowered the temperature enough to allow the
    primers, which were small and in VAST EXCESS, to
    bind (ANNEAL) to their respective complementary
    target DNA base pair-sequences.
  • At this point he added DNA polymerase and allowed
    the polymerization reaction with the triphosphate
    nucleotides to occur.
  • That is, the DNA polymerase FILLED IN the missing
    portion of each strand making TWO NEW DOUBLE
    STRANDED regions of DNA

10
(No Transcript)
11
Reverse Transcriptase cDNAcomplementary DNA
  • Bacteria is used to make large quanities of
    eukaryotic genes and protiens
  • Eukaryote have introns and exons
  • Prokaryotes do not
  • The trick is to modify the gene you want into a
    gene the prokaryotes can transcibe
  • Reverse transcriptase

12
(No Transcript)
13
(No Transcript)
14
(No Transcript)
15
(No Transcript)
16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
Synthetic Oligonucleotides
  • Selectively block either the 3 end or the 5 end
  • Ensures that the condensation reactions forming
    the sugar phosphate bond occur in the proper
    sequence for the desired order

20
(No Transcript)
21
Antisense oligonucleotides are synthetic
fragments of ribo or deoxyribonucleic acids which
specifically binds to their complementary
messenger RNA and then, block the corresponding
protein translation. 
22
Plasmids as Vector
  • Restriction enzymes are used
  • Ex
  • EcoRI
  • Useful as vectors b/c multiply rapidly and are
    easily taken up by bacteria through cell membrane
  • Multiple copies are called clone

Use of plamids Insulin and Human GH
23
Radioactive Probes
  • Used to isolate DNA or mRNA
  • To do this application of nucleic acid
    hybridzation, using radioactive probes
  • Probes short segment of single-stranded DNA or
    RNA labeled with a radioactive isotope

24
(No Transcript)
25
(No Transcript)
26
(No Transcript)
27
DNA Sequencing
28
(No Transcript)
Write a Comment
User Comments (0)
About PowerShow.com