Module 2 - Genetic techniques Flashcards by Amanda Harnett

How can knowing about the DNA sequence/structure help us?

Used for

producing drugs, vaccines
identifying if you have a genetic disease
helping to cure genetic disease
forensics
genetic screening and DNA testing
genetic counselling
Research

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PCR

Polymerase Chain Reaction

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PCR Allows specific region of DNA to be —————- exponentially from minute quantity templates

Amplified

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PCR Offers alternative approach to —- ——- for the production of many copies of identical sequence of DNA

Gene cloning

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IN PCR starting material may be genomic —, —, ————– ————- DNA, ——– DNA or ————- —————

DNA
RNA
Archival specimen
Cloned
Forensic samples

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enzymes which can make DNA from dNTPs provided a template or primer is available to start the process

Polymerase

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First step in PCR

denaturation of double strand of DNA

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Second step in PCR

annealing of primers

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Third step in PCR

elongation of strands

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cDNA

Copy of DNA

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Denaturation of dsDNA - typically temp used is ————— for human genomic DNA.

93-95 degrees C

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Annealing - temp ————-, depending on melting temperature of the expected duplex.

40-70 degrees C

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DNA Synthesis - generally ———–: use the optimum temperature for the polymerase being used.

70-75 degrees C

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Product size = Length of ————–/—————/left ————– + Length of ———–/———————–/right —————- + —————- to be amplified

Forward, upstream, primer
Reverse, downstream, primer
Region

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PCR:
DNA is ————–
————Attach to each strand
A new DNA strand is ——————– ————- primers on each template strand

denatured
Primers
Synthesised
Behind

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Continued rounds of —————–swiftly produce large numbers of identical fragments

Amplification

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Applications of PCR: D———— and S————- of genetic diseases and cancer

Diagnosis; Screening

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Applications of PCR: Rapid detection of slowly growing ————————-

Microorganisms

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Applications of PCR: — typing in T———————–

HLA; transplantation

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Applications of PCR: Analysis of DNA in A———– M————-

Archival material

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Applications of PCR: DNA F——————— in forensic science

Fingerprinting

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Applications of PCR: Preparation of nucleic acid P————-

Probes

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Applications of PCR: ———— screening, mapping and subcloning

Clone

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detection of mRNA expression levels

RT-PCR

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RT-PCR traditionally involves ----- steps

two

Step 1. the ------ reaction and ----------- amplification. RNA is first reverse transcribed into --------- using a reverse ---------------------

RT; PCR; cDNA; transcriptase

Step 2. the resulting ----- is used as templates for subsequent ------------- using ------------ specific for one or more genes.

cDNA; PCR amplification; primers

Genetic Techniques 1

Diagnostic PCR- template DNA

Genetic Techniques 2

RT-PCR – template RNA, can be diagnostic

Genetic Techniques 3

DNA sequencing

Process of manipulating genes, usually outside the organisms normal reproductive process.

Genetic engineering

Genetic engineering Usually involves -----------, -------------------- and ------------------ of DNA into cells or model organisms usually to express a ---------------

isolation; manipulation; reintroduction; protein

Genetic Engineering Techniques: 3 examples

Recombinant DNA, cloning gene therapy

What two things are genetic engineering techniques used for?

production of drugs, vaccines, treatment strategies | research e.g. investigating mechanisms of disease

Recombinant DNA technology: Basic procedure 1: DNA ----------- from cells or tissues

purified

Recombinant DNA technology: Basic procedure 2: ------------ ---------------- used to generate specific DNA fragments, recognise and cut DNA at specific sequence

Restriction enzymes

Recombinant DNA technology: Basic procedure 3: Fragments join to other DNA molecules which act as ------------- (carrier molecules), ---------------- + DNA fragment= --------------- DNA molecule

vectors; vector; recombinant

Recombinant DNA technology: Basic procedure 4: Recombinant DNA molecule transferred to ------- ---------

host cell

Recombinant DNA technology: Basic procedure 5: Within host cell recombinant --------------- producing dozens of identical copies

replicates

Recombinant DNA -As host replicates, recombinant molecule passed to all -------------- creating population of cells each carrying cloned DNA sequence

progeny

Recombinant DNA technology: Basic procedure 7: Cloned DNA can be recovered from host cells, ----------- and ---------------

purified; analysed

Recombinant DNA technology: Basic procedure 8: Cloned DNA in host cells is ------------------, its mRNA ----------------, gene product ------------- and used or sold

transcribed; translated; isolated

First genetically engineered drug =

human insulin (1982)

First genetically engineered vaccine =

hepatitis B (1986)

the creation of an exact genetic replica of a small segment of DNA, a cell or a whole organism

Reproductive cloning

stem cells

Therapeutic cloning

Cloning = ---------------- ------------- cell + egg cell with --------------- removed

Somatic body; Nucleus

Nucleus of somatic body cell fused with denucelated egg cell

cloning

reproductive cloning - ---------------- mother

surrogate

Therapeutic cloning ---> ------------ --------------------

Tissue culture

Dolly the sheep - Cloned in ----- - Died in ------------

1997; 2003

a technique for correcting defective genes responsible for disease development

Gene Therapy

In most cases of gene therapy, a “normal” gene is ------------- into the genome to ------------- an “abnormal” disease causing gene.

inserted; replace

There are two major techniques in gene therapy

Gene Augmentation Therapy | Targeted Modulation of gene expression

The introduction of DNA into a cell with a view to augmenting gene function, e.g. recessive diseases.

Gene Augmentation Therapy

could also be used to deliver genes encoding proteins that would be responsible for stimulating the immune system to treat cancer or infections

Gene Augmentation Therapy

Methods for turning off genes using for example ribozymes and anti-sense oligonucleotides, e.g., infectious disease and cancers (later lecture).

Targeted Modulation of gene expression

A ----------- needed to deliver the therapeutic gene to the patients target cells

vector

Gene therapy: Most common vector is -------

virus

First human gene therapy experiment when?

1990

Gene therapy is not really successful in humans due to the lack of efficient ---------- ------------- for gene delivery.

vector systems

DNA delivered to cells can be ----------- and in the case of cystic fibrosis restore ---------------

expressed; function

Gene therapy: Shift in emphasis to ---------- and -------- rather than ‘genetic diseases

Cancer; HIV

DNA testing 1: collect a persons --------------- ---------

total DNA

DNA testing 2: DNA cut into small pieces with -------------- that are chemical scissors

enzymes

DNA testing 3: DNA fragments put on ------

gel

DNA testing 4: fragments separated according to ----------- using an ----------- -----------

size; electrical current

DNA testing 5: Separated fragments transferred to ------------ ----------------- and mixed with DNA ----------- that picks up the regions of DNA interest

nylon membrane; probe

Provides information and support to an individual or family about genetic disorders

Genetic Counselling

Who uses Genetic Counselling?

A child is born with several abnormalities There is a history in the family of a medical condition A woman has a history of several miscarriages Woman aged over 35 yrs and pregnant Anyone who has uncertainties about genetic risks

the examination of a person’s chromosomes, DNA or the biochemical product of a gene (protein)

Genetic Testing

Tissues tested in genetic testing:

blood; skin; saliva; hair follicles; amniotic; CVS; placental

CVS test done in pregnancy around ------ weeks

11-12

Amniocentesis done in pregnancy around -------- weeks

15-19

CVS = -------------- tissue withdrawn

chorionic

Amneocentesis = ------------- -------------- withdrawn

amniotic fluid

3 types of Genetic Tests Used

Cytogenic testing - examine chromosomes DNA testing – DNA extracted from cells of a variety of body fluids or tissues Proteins – usually tested for in blood

Research: Genetic techniques used all the time to ------------------- ----------------

investigate diseases

Research: Identify ---------- and ------------- ----------------

therapeutic; diagnostic markers

Research: Understand ------------ ------------- involved in development of diseases

molecular mechanisms

Research: Identify genes that are ------------------- and cause disease

abnormal