19.1 Techniques of Molecular Genetics Have Revolutionized Biology

1
19.1 Techniques of Molecular Genetics Have
Revolutionized Biology

• Recombinant DNA TechnologyGenetic
  EngineeringBiotechnology
• Locating, isolating, altering, and studying DNA
  segments
• Biotechnology
• Using recombinant DNA technology to develop new
  biological products

2
19.2 Molecular Techniques Are Used to Isolate,
Recombine, and Amplify Genes
3
Cutting and Joining DNA Fragments

• Restriction enzymes recognizing and cutting DNA
  at specific nucleotide sequences
• Palindromic sequences
• Immune system of bacteria
• Type II restriction enzyme most useful enzyme
• By adding methyl groups to the recognition
  sequence to protect itself from being digested by
  its own enzyme in bacteria

4
(No Transcript)
5
Cutting and Joining DNA Fragments

• Cohesive ends fragments with short,
  single-stranded overhanging ends
• Blunt ends even-length ends from both single
  strands

6
(No Transcript)
7
(No Transcript)
8
Viewing DNA Fragments

• Gel electrophoresis-separation of DNA fragments
  by size through a gel medium
• Smaller fragments migrate faster

9
(No Transcript)
10
(No Transcript)
11
Viewing DNA Fragments

• Locating DNA fragments with Southern blotting and
  probes
• Probe DNA or RNA with a base sequence
  complementary to a sequence in the gene of
  interest
• Is usually labeled for easy detection
• Radioactive P32
• Fluorescent tag

12
(No Transcript)
13
Cloning Genes

• Gene cloning amplifying a specific piece of DNA
  via a bacteria cell
• Cloning vector a replicating DNA molecule
  attached with a foreign DNA fragment to be
  introduced into a cell
• Has features that make it easier to insert DNA
  and select for presence of vector in cell.
• Origin of replication
• Antibiotic resistance gene
• Cloning site

14
(No Transcript)
15
Cloning Genes

• Plasmid vectors
• Linkers synthetic DNA fragments containing
  restriction sites
• Transformation of host cells with plasmids
• Selectable markers are used to confirm whether
  the cells have been transformed or not.

16
(No Transcript)
17
(No Transcript)
18
(No Transcript)
19
(No Transcript)
20
(No Transcript)
21
Amplifying DNA fragments with the polymerase
chain reaction (PCR)

• Taq polymerase stable DNA polymerase at high
  temperature
• Researcher designs specific oligonucleotide
  primers that serve as the ends of the amplified
  fragment
• Very similar to replication

22
(No Transcript)
23
(No Transcript)
24
19.3 Molecular Techniques Can Be Used to Find
Genes of Interest
25
Gene Libraries

• Gene library a collection of clones containing
  all the DNA fragments from one source
• Creating a genomic DNA library
• cDNA library consisting only of those DNA
  sequences that are transcribed into mRNA
• Creating a cDNA library

26
(No Transcript)
27
(No Transcript)
28
Gene Libraries

• Screening DNA libraries
• Plating clones of the library
• Probing plated colonies or plaques

29
(No Transcript)
30
In Situ Hybridization

• DNA probes used to determine the chromosomal
  location and to visualize a gene while it is in a
  cell
• May also be used to detect localization of mRNA
  expression in multicellular organisms.

31
(No Transcript)
32
(No Transcript)
33

• Positional Cloning
• Isolating genes on the basis of their position on
  a genetic map

34
(No Transcript)
35
19.4 DNA Sequences Can Be Determined and Analyzed

• Restriction Fragment Length Polymorphisms (RFLPs)
• Some DNA fragments have different restriction
  sites due to mutation for the same restriction
  enzyme, which causes polymorphisms within a
  population.

36
(No Transcript)
37
DNA Sequencing

• Sangers dideoxy-sequencing method
• Dideoxyribonucleoside triphosphate (ddNTP) lacks
  a 3'-oh group, which terminates DNA synthesis.

38
(No Transcript)
39
(No Transcript)
40
(No Transcript)
41
DNA Fingerprinting (DNA Profiling)

• Microsatellites variable number of copies of
  repeat sequences possessed by many organisms,
  which can be amplified by PCR
• Combined with RFLP analysis to form more
  thorough fingerprint

42
(No Transcript)
43
19.5 Molecular Techniques Are Increasingly Used
to Analyze Gene Function

• Forward genetics
• Begins with a phenotype to a gene that encodes
  the phenotype
• Reverse genetics
• Begins with a gene of unknown function, first
  inducing mutations and then checking the effect
  of the mutation on the phenotype

44
19.5 Molecular Techniques Are Increasingly Used
to Analyze Gene Function

• Site-directed mutagenesis
• Reverse genetics creating mutation in particular
  DNA sequences, and then studying the effects of
  these mutation on the organisms

45
19.5 Molecular Techniques Are Increasingly Used
to Analyze Gene Function

• Silencing genes with RNAi
• Using RNAi for the treatment of human disease
  lowering ApoB with RNAi
• Model Genetic Organism
• The mouse, Mus musculus

46
(No Transcript)
47
19.6 Biotechnology Harnesses the Power of
Molecular Genetics

• Pharmaceuticals
• Human insulin
• Specialized bacteria
• Agricultural products
• Oligo nucleotide drugs
• Genetic testing
• Gene therapy
• Direct transfer of genes into humans to treat
  disease