• How do restriction enzymes and gel electrophoresis allow scientists to analyze DNA and perform genetic testing?
The Tools of Molecular Biology – Scientists use different techniques to: • • • • •
extract DNA from cells cut DNA into smaller pieces identify the sequence of bases in a DNA molecule make unlimited copies of DNA Genetically engineer organisms
The Tools of Molecular Biology
The Tools of Molecular Biology – DNA Extraction • DNA can be extracted from most cells by a simple chemical procedure. • The cells are opened and the DNA is separated from the other cell parts.
The Tools of Molecular Biology
– Cutting DNA with restriction enzymes • DNA molecules (chromosomes)are too large to be analyzed • restriction enzymes cut DNA into smaller fragments at specific sites.
Recognition sequences
DNA sequence
Restriction enzyme EcoR I cuts the DNA into fragments
Sticky end
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Figure 20.10a
Normal -globin allele
• Restriction Enzymes can be used to identify mutations in specific alleles
175 bp
Large fragment
201 bp
DdeI
DdeI
DdeI
DdeI
Sickle-cell mutant -globin allele Large fragment
376 bp
DdeI
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene
Figure 20.10b
Figure 20.11
TECHNIQUE
Normal allele
Sickle-cell allele
DNA restriction enzyme
Restriction fragments
I
II III
Heavy weight
Nitrocellulose membrane (blot) Gel Sponge
Large fragment
I Normal II Sickle-cell III Heterozygote -globin allele allele 1 Preparation of restriction fragments
Alkaline solution 2 Gel electrophoresis
Paper towels 3 DNA transfer (blotting)
376 bp 201 bp 175 bp (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles
I
II III
Radioactively labeled probe for -globin gene
Nitrocellulose blot
Probe base-pairs with fragments Fragment from sickle-cell -globin allele Fragment from normal - globin allele
I
II III
Film over blot 5 Probe detection
4 Hybridization with labeled probe
– Separating DNA fragments
• Why are restriction enzymes useful and how do they work?
• In gel electrophoresis, DNA fragments are placed at one end of a porous gel, and an electric voltage is applied to the gel. • The negatively charged DNA molecules move toward the positive end of the gel. • DNA fragments separate by size, smaller pieces travelling farther.
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Gel Electrophoresis • What causes the DNA to move down the gel? • How does the gel help separate the various DNA pieces?
Power source
DNA plus restriction enzyme
Longer fragments
Mixture of DNA fragments
Gel
Shorter fragments Negatively charged phosphate groups attracted to positive ple
Gel Electrophoresis
Gel Electrophoresis Lab • We have a sample of DNA found at a crime scene • We also have DNA samples from two suspects • You will work as a table group and: – – – – –
• Gel electrophoresis can be used to compare the genomes of different species or individuals within a species. • It can also be used to isolate one particular gene in an individual's genome.
Share one of the gels with two other table groups Load 18 uL of DNA from each sample into one lane on the gel Use a new tip every sample Write down which gel you used and which lanes you used Use only lanes 1,2,3 or 5,6,7, or 10, 11, 12
DNA Forensics
Genome: the entirety of an organisms heredity information For a Human: 3 Billion base pairs 1.5% are genes coding for proteins Exons (1.5%)
Regulatory sequences (20%)
Repetitive DNA that includes transposable elements and related sequences (44%) L1 sequences (17%)
Alu elements (10%)
Introns (5%)
• Because of random mutations in DNA, cutting different people’s genomes with same restriction enzymes produces different sized pieces of DNA, creating a “DNA fingerprint”
Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (14%)
Simple sequence DNA (3%)
Large-segment duplications (56%)
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Crime Scene Analysis • Why should everyone’s DNA fingerprint be unique?
• On a separate piece of paper, include the following: – Record a data table that includes the number of bands for each lane on your gel and the distance (in millimeters) that each band travelled from the well – Write a brief conclusion indicating which if any of the suspects DNA fingerprint matches the fingerprint of the DNA found at the crime scene. Explain how you know which one matches and how you know that the other does not match. Distance Travelled in mm Crime Scene Fragment 1
Suspect #1
Suspect #2
15 mm
Fragment 2
Essential Question
PCR: Polymerase Chain Reaction
• How do PCR and DNA sequencing allow scientists to use the mechanics of DNA replication to identify alleles?
– Making Copies • Polymerase chain reaction (PCR) allows scientists to make many copies of genes. • -Virtual Lab
Figure 20.10a
DNA Sequencing
Normal -globin allele 175 bp
DdeI
• Knowing the DNA Sequence Allows researchers to:
Large fragment
201 bp
DdeI
DdeI
DdeI
Sickle-cell mutant -globin allele Large fragment
376 bp
DdeI
• Compare genes between organisms • Discover functions of different genes and gene combinations. • Identify genes that cause genetic disorders
DdeI
DdeI
(a) DdeI restriction sites in normal and sickle-cell alleles of the -globin gene
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DNA Sequencing
DNA Sequencing – Reading the Sequence • In DNA sequencing, a complementary DNA strand is made using a small proportion of fluorescently labeled nucleotides.
Dye molecules
DNA Sequencing
Essential Question • What technology options are available for human application?
Gene Therapy
Available Technology • Sperm selection/sex determination (carrying X or Y chromosome) • Pre-implantation diagnosis with in-vitro fertilization prior to embryo implantation • Testing of fetus during pregnancy: Amniocentesis and CVS • Gene Therapy- insert functional gene to replace defective gene – into egg prior to fertilization or post-birth
