Lecture ONE: Foundation Course Genetics Tools of Human Molecular Genetics I

Lecture ONE: Lecture ONE: Foundation Course GeneticsFoundation Course Genetics

Tools of Human Molecular Genetics ITools of Human Molecular Genetics I

Discuss the following:Discuss the following:

• Genetic engineering

• Molecular Genetics

• Genetic Screening

• Recombinant DNA

• Stem cells

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Copyright © 2005 Brooks/Cole — Thomson Learning

Biology, Seventh Edition CHAPTER 14 DNA Technologies

Recombinant DNA methods• Restriction enzymes

–Enzymes from bacteria–Used to cut DNA molecules in specific places

• cut DNA is placed in a Vector carrier • DNA ligase joins cut pieces of DNA

• Transformation: uptake of foreign DNA into cells

Cloning Vectors

Vector Maximum Insert size

Approx. No. of clones required in library

plasmid 10 kb 10 x 105

lambda 20 kb 5 x 105

cosmid 45 kb 2 x 105

YAC 1 Mb 104

BAC > 500 kb 5 x 104

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Plasmids

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Cutting DNA with a restriction enzyme

• Restriction enzymes are molecular scissors which cut DNA at specific base sequences.

E.g. HindIII and EcoR1 are restriction enzymes

• Hind III cuts DNA at the recognition sequence 5’-AAGCTT-3’

• EcoR1 cuts DNA at the recognition sequence 5’- GAATTC-3’

Restriction Enzymes fig 14.2Restriction Enzymes fig 14.2

Restriction Enzymes: (Fig 14-1)Restriction Enzymes: (Fig 14-1)

• Many of the sites in DNA which restriction enzymes recognize are

PALINDROMIC

• Palindromic: means that the sequence reads the same 5’-3’ in both directions

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Cutting DNA with a restriction enzyme

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• Splicing foreign DNA into a vector• Foreign DNA and plasmid DNA cut with

same restriction enzyme• Produces linear molecules with

complementary single-stranded ends• Recombinant DNA created by mixing so

sticky ends pair• DNA ligase forms covalent bonds, linking

the two fragments

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Producing a genomic or chromosome library

• Genomic library• Collection of DNA fragments that

represent all the DNA in the genome

• Chromosome library• All the DNA fragments in that specific

chromosome

• cDNA library• Produced using reverse transcriptase• Makes DNA copies of mature mRNA

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Cloning DNACloning DNA: :

• The recombinant DNA can be put back into bacteria and the bacteria allowed to grow

• This will produce many genetically identical copies of the piece of DNA. This is called cloning

• A clone is a genetically identical individual or cell

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• Genetic probes• Segments of single-stranded DNA that

can hybridize to complementary base sequences in target gene

• Southern blot technique

Southern Northern, and Western Southern Northern, and Western BlottingBlotting

•Southern Blotting: DNA

•Northern Blotting: RNA

•Western Blotting : Protein

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Using a geneticprobe to find bacterial cellswith a specificrecombinant DNA molecule

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• Amplifying DNA in vitro by PCR–Small amount of double-stranded DNA–DNA precursors–Specific nucleic acid primers–Taq DNA polymerase

• DNA is denatured• Primers attach to primer-binding site on

each DNA strand• Each strand acts as template for DNA

synthesis

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Amplification of DNA by PCR

Types of PCR ITypes of PCR I

RT PCR Reverses transcriptase PCR. DNA is amplified

from mRNA.

QRT PCR Quantitative Real Time PCR the amplified product is linked to a fluorescent

reporter molecule, the fluorescence is measured at each cycle. This allows the amplification to be monitored to optimize the efficiency of amplification.

Types of PCR IITypes of PCR II

Multiplex PCR two or more sets of primers are used in

the same reaction mix.

Nested PCR (also nested RT-PCR) There are two amplifications made: the:

The first amplifying a large product which, in the second amplification is used as the template.

Gel ElectrophoresisGel Electrophoresis

• A method of separation pieces of DNA

• Horizontal gel (Agarose)

• Vertical (Polyacrylamide)

• Many variations on the methods exist

The DNA is negatively charged and migrates towards the positive pole

The smallest fragments move the fastest through the gel and therefore move the furthestThe largest fragments move the least By comparing the fragments in the sample with standards of known sizethe size of the sample fragments can be estimated

Links to virtual labsLinks to virtual labs

• DNA extractionhttp://learn.genetics.utah.edu/units/biotech/extraction

/

• Making a gelhttp://learn.genetics.utah.edu

/units/biotech/gel

http://learn.genetics.utah.edu/units/biotech/extraction/



http://learn.genetics.utah.edu/units/biotech/gel



Stem Cells are undifferentiated cellsStem Cells are undifferentiated cells

Type of Stem Cell Developmental Potential

Early Embryonic Totipotent

Blastocyst Embryonic Pluripotent

Fetal Pluripotent

Umbilical Multipotent

Adult Multipotenthttp://learn.genetics.utah.edu/units/stemcells

http://learn.genetics.utah.edu/units/stemcells

http://learn.genetics.utah.edu/units/stemcells

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Possible therapeutic uses of stem cellsPossible therapeutic uses of stem cells

• Treatment of disease • Diabetes

• Parkinsons disease

• Treatment of spinal injury

• Culture of differentiated tissues

Documents

Lecture ONE: Foundation Course Genetics Tools of Human Molecular Genetics I