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Essential Question

Not enough data for L to know if she is a carrier

• 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 (56%)

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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

• Gene Testing – – – – – –

Preimplantation Prenatal Newborn Diagnostic Carrier Predictive/Presymptomatic

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Figure 47.12

1 Blastocyst reaches uterus. Uterus

Endometrial epithelium (uterine lining) Inner cell mass Trophoblast

Gene Therapy

Blastocoel

2 Blastocyst implants (7 days after fertilization).

Expanding region of trophoblast Maternal blood vessel

IVF Website

Epiblast Hypoblast Trophoblast

Gene Testing Site 3 Extraembryonic membranes start to form (10–11 days), and gastrulation begins (13 days).

http://www.ncbi.nlm.nih.gov/gtr/

Expanding region of trophoblast Amniotic cavity Epiblast Hypoblast Yolk sac (from hypoblast) Extraembryonic mesoderm cells (from epiblast) Chorion (from trophoblast)

4 Gastrulation has produced a three-layered embryo with four extraembryonic membranes.

Amnion Chorion Ectoderm Mesoderm Endoderm Yolk sac Extraembryonic mesoderm Allantois

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