DNA TECH

1
DNA TECH

• Review for Lecture P161

2
most important concept

•      
• DNA -gtmore DNA --gt RNA -gt protein
• This "central dogma" of today's molecular
  biology has applications
• in medicine and biotechnology.

3
APPLICATIONS

•   
• IDENTIFYING
• genetic biodiversity
• genetic disorders
• corpses and suspects
• and babies switched at birth.

4
Genetic engineering and gene
therapy are other applications of the "central
dogma" in which we create different (better?)
proteins by changing the DNA.

• Phenotypes are produced by
• proteins
• products of chemical reactions catalyzed by
  proteins
• regulation of the timing and location (which
  cells in which organs?) of replication,
  transcription, and translation.

5
DNA -gtmore DNA --gt RNA -gt protein

• Francis Crick said that
• DNA makes RNA
• RNA makes protein
• and protein makes us.

6
Proteins make us?

• Phenotypes R us?
• Phenotypes are produced by
• proteins
• products of chemical reactions catalyzed by
  proteins
• regulation of the timing and location (which
  cells in which organs?) of replication,
  transcription, and translation.

7
APPLICATIONS

•   
• Can we change phenotypes by changing the
  genotype, the DNA?
• Should we?
• Who should decide?
• Lab 11 (6 weeks?)

8
Biotech TOOLS

• restriction endonuclease enzymes
• nucleic acid fragments (RFLPs, etc.)
• electrophoresis
• probes
• PCR
• sequencing
• ligase enzyme
• synthetic nucleotides
• more later

9
Biotech TOOLS

•   For each, know
• what it does
• why anybody would use it
• how or why it works especially if it involves
  something relevant to the natural structure and
  function of DNA.

10
1. Restriction enzymes

• Restriction enzymes are nucleases they cut DNA
  or RNA into fragments.
• Restriction enzymes are used to create cuts and
  fragments for DNA analysis and also for genetic
  engineering.

11
(No Transcript)
12
(No Transcript)
13
(No Transcript)
14
( Box 15.1, p.302).
15
1. Restriction enzymes

• Restriction enzymes are nucleases they cut DNA
  or RNA into fragments.
• Restriction enzymes are used to create cuts and
  fragments for DNA analysis and also for genetic
  engineering.

16
2. Fragments

• Fragments produced by restriction enzymes have
  different lengths. Fragments are sometimes called
  RFLPs especially when the different lengths
  reflect differing alleles or at least individual
  differences.
• Uses next

17
2. Fragment Uses

• Restriction enzymes are used to create cuts and
  fragments for DNA analysis, sequencing, and also
  for genetic engineering.

18
3. Electrophoresis

• Electrophoresis separates and sorts fragments
  according to their lengths

19
(No Transcript)
20
(No Transcript)
21

• Box 3.2

22
electrophoresis

• Screeningfordisorders

23
3. electrophoresis

• Some DNA uses separating fragments for
• screening for disorders
• ID or clear suspects
• sequencing
• and more

24
4. PROBES

• Probes are oligonucleotides, short pieces of RNA
  or single-stranded DNA. Probes work by
  hybridizing with single strands of DNA or RNA
  it's called hybrid (new meaning) because the
  probe and its target are from different critters.

25
4. PROBES

• The probe and its target have complementary base
  sequences so they stick together by hydrogen
  bonds between the complementary pairs, just like
  a primer will stick to DNA in replication or
  transcription. Radioactive or fluorescent probes
  are especially useful in research. Can you figure
  out why?
• .

26
4. PROBES

• A probe can ID specific base sequences on the
  electrophoresis gel or on blots (box 15.2, p.
  309) which absorb the fragments from the gel.
• .

27
(No Transcript)
28
(No Transcript)
29
(No Transcript)
30
4. PROBES

• The probe and its target have complementary base
  sequences so they stick together by hydrogen
  bonds between the complementary pairs
• A probe can also pinpoint the location of a gene
  on a chromosome (as you will see in CD activity
  16.1)
• A probe can find which colonies of genetically
  engineered bacteria have the right DNA sequence
  (as you will see in CD activity 17.1).
• .

31
4. PROBES

• Probes can also be used in vivo one example is
  in situ hybridization which is used to tell which
  genes are active in specific tissues. The mRNAs
  which have been transcribed can be detected with
  radioactive or fluorescent DNA probes (Box 19.1).
  

32
(No Transcript)
33
5. PCR

• The polymerase chain reaction, fig. 12.11, uses
  primers and polymerase and temperature cycles to
  replicate DNA in huge quantities.
• PCR DNA copy-making study pp. 240b-242a
  (especially fig. 12.11a, 12.11b, 12.11b continued
  ) and CD activity 12.2.

34
(No Transcript)
35
(No Transcript)
36
(No Transcript)
37
6. SEQUENCING

• determining the sequence of bases in DNA
• The Sanger (dideoxy) procedure is still used in
  some professional laboratories today

38
dNTP deoxynucleotide triphosphate ddNTP di -
deoxy NTP
39
(No Transcript)
40
(No Transcript)
41
Newer methods (chapter 16)

• Dyes on different nucleotides instead of
  radioactivity
• (still electrophoresis, but no longer 4 bands)
• machine scanner tabulates color sequences
  (instead of error-prone human looking at film)

42
(No Transcript)
43
(No Transcript)
44
sequencing machines
45
7. LIGASE

• Ligase enzyme can make recombinant DNA for
  genetic engineering or GM (genetic modification).
  Ligase "glues" fragments together, usually into
  circles of DNA, called plasmids (which are
  natural) or BACs (bacterial artificial
  chromosomes).

46
(No Transcript)
47
Recombinant DNA is not expressed (cannot work)
unless it is adjacent to promoter DNA segments
48
In live normal cells, the natural function of
ligase is to combine the Okazaki fragments of the
lagging strand in DNA replication

49
8. Synthetic Nucleotides

• DNA can be synthesized in the lab, but synthetic
  DNA is used mostly as probes or sometimes as
  deliberate mutations, tiny inserts into
  recombinant DNA. You don't have to know how they
  synthesize DNA. You can buy synthetic DNA and
  RNA.

50
DNA used for genetic engineering is
always recombinant DNA, involving genes from
natural sources

• partly because real genes are too long to be
  synthesized practically
• and partly because the proteins which would be
  translated do not fold into functional and
  predictable tertiary shapes. Scientists don't
  know enough about proteins yet. We still need
  cells.

51
most important concept

•      DNA -gtmore DNA --gt RNA -gt protein
• the natural processes of replication can be used
  in the laboratory to synthesize bits of DNA for
  analyzing more DNA--
• disorders,
• forensics,
• archaeology and paleontology,
• pure research on how cells work....

52
(No Transcript)